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pubmed23n0001_1100 | Bacterial and fungal growth in total parenteral nutrition solutions,. | The most serious complication of prolonged intravenous infusion of hypertonic dextrose and amino acids is infection. Frequently, the etiology is fungal rather than bacterial. Previous authors have suggested that bacterial survival and growth in the solutions is suppressed by (a) high dextrose concentration, (b) high osmolality, or (c) low pH. This paper presents evidence that proposals (a) and (b) are untenable and (c) is only partly responsible. We call attention to the presence of a factor that is antibacterial but not antifungal; namely, a high concentration of glycine. | Bacterial and fungal growth in total parenteral nutrition solutions,. The most serious complication of prolonged intravenous infusion of hypertonic dextrose and amino acids is infection. Frequently, the etiology is fungal rather than bacterial. Previous authors have suggested that bacterial survival and growth in the solutions is suppressed by (a) high dextrose concentration, (b) high osmolality, or (c) low pH. This paper presents evidence that proposals (a) and (b) are untenable and (c) is only partly responsible. We call attention to the presence of a factor that is antibacterial but not antifungal; namely, a high concentration of glycine. | 2,102 |
pubmed23n0001_1101 | New medium for isolating iron-oxidizing and heterotrophic acidophilic bacteria from acid mine drainage. | A new solid medium is described for growing iron and heterotrophic bacteria from acid mine drainage (AMD). Examination of AMD from five states revealed several kinds of colonies of iron-oxidizing bacteria: (i) smooth, (ii) smooth with secondary growth sectors or branching, (iii) star-shaped, (iv) radiating lobe, and (v) flat-rough. All AMD samples yielded whitish colonies that could not use ferrous iron, sulfur, or hydrogen, nor could they grow on nutrient agar, brain heart infusion agar, or Trypticase soy agar. Glucose and sucrose supported growth if the sugar-salts medium was at pH 3.0. The new iron medium has several advantages over others: (i) easy preparation, (ii) rapid growth, (iii) larger colonies, (iv) differentiation of colony morphology, and (v) detection of a new group of heterotrophic acidophilic bacteria. | New medium for isolating iron-oxidizing and heterotrophic acidophilic bacteria from acid mine drainage. A new solid medium is described for growing iron and heterotrophic bacteria from acid mine drainage (AMD). Examination of AMD from five states revealed several kinds of colonies of iron-oxidizing bacteria: (i) smooth, (ii) smooth with secondary growth sectors or branching, (iii) star-shaped, (iv) radiating lobe, and (v) flat-rough. All AMD samples yielded whitish colonies that could not use ferrous iron, sulfur, or hydrogen, nor could they grow on nutrient agar, brain heart infusion agar, or Trypticase soy agar. Glucose and sucrose supported growth if the sugar-salts medium was at pH 3.0. The new iron medium has several advantages over others: (i) easy preparation, (ii) rapid growth, (iii) larger colonies, (iv) differentiation of colony morphology, and (v) detection of a new group of heterotrophic acidophilic bacteria. | 2,103 |
pubmed23n0001_1102 | Effect of initial pH on aflatoxin production. | The effect of initial pH on aflatoxin production by Aspergillus parasiticus NRRL 2999 was examined in a semisynthetic medium. Maximal growth, aflatoxin production, and aflatoxin production per unit of growth occurred at initial pH levels of 5.0, 6.0, and 7.0 respectively. Initial pH levels less than pH 6.0 favored production of the B toxins, whereas levels greater than pH 6.0 favored production of the G toxins. | Effect of initial pH on aflatoxin production. The effect of initial pH on aflatoxin production by Aspergillus parasiticus NRRL 2999 was examined in a semisynthetic medium. Maximal growth, aflatoxin production, and aflatoxin production per unit of growth occurred at initial pH levels of 5.0, 6.0, and 7.0 respectively. Initial pH levels less than pH 6.0 favored production of the B toxins, whereas levels greater than pH 6.0 favored production of the G toxins. | 2,104 |
pubmed23n0001_1103 | High-temperature production of protein-enriched feed from cassava by fungi. | A simple, nonaseptic, low-cast process for the conversion of cassava, a starchy tropical root crop, into microbial protein for use as animal feed was sought. Screening tests culminated in the isolation of a thermotolerant, amylase-producing mold, designated I-21, which was identified as Aspergillus fumigatus. The optimum pH for protein synthesis was 3-5, but the optimum temperature was less than the desired temperature (larger than or equal to 45 C) required for a nonaseptic fermentation. A. fumigatus I-21 and its asporogenous mutant I-21A grew equally well in a medium prepared from whole cassava roots with a mean protein doubling time at 45 C and pH 3.5 of 3.5 h. In batch culture, approximately 4% carbohydrate, supplied as whole cassava, could be feremented in 20 h, giving a final yield of 24 g of dry product, containing 36.9% crude protein, per liter. The conversion of carbohydrate used to crude protein was 22.1%. When determined as amino acids, the protein content of the product, which contained cassava bark and other unfermented residues, was 27.1%. With urea as the nitrogen source, no pH control was necessary. Preliminary data indicated that medium prepared from whole cassava roots was inhibitory to the mold unless the cassava pulp was heated to 70 C immediately after being ground. Heating to 70 C was required to gelatinize the starch and permit its complete utilization. | High-temperature production of protein-enriched feed from cassava by fungi. A simple, nonaseptic, low-cast process for the conversion of cassava, a starchy tropical root crop, into microbial protein for use as animal feed was sought. Screening tests culminated in the isolation of a thermotolerant, amylase-producing mold, designated I-21, which was identified as Aspergillus fumigatus. The optimum pH for protein synthesis was 3-5, but the optimum temperature was less than the desired temperature (larger than or equal to 45 C) required for a nonaseptic fermentation. A. fumigatus I-21 and its asporogenous mutant I-21A grew equally well in a medium prepared from whole cassava roots with a mean protein doubling time at 45 C and pH 3.5 of 3.5 h. In batch culture, approximately 4% carbohydrate, supplied as whole cassava, could be feremented in 20 h, giving a final yield of 24 g of dry product, containing 36.9% crude protein, per liter. The conversion of carbohydrate used to crude protein was 22.1%. When determined as amino acids, the protein content of the product, which contained cassava bark and other unfermented residues, was 27.1%. With urea as the nitrogen source, no pH control was necessary. Preliminary data indicated that medium prepared from whole cassava roots was inhibitory to the mold unless the cassava pulp was heated to 70 C immediately after being ground. Heating to 70 C was required to gelatinize the starch and permit its complete utilization. | 2,105 |
pubmed23n0001_1104 | Removal of algae from Florida lakes by magnetic filtration. | Magnetic filtration was used for the removal of algal populations present in five lakes located in the vicinity of Gainesville, Fla. It was found that the use of this technique enabled a good removal (94%) of algal cells from three lakes where the pH was around 7. The other two lakes, with a higher pH, displayed a lower removal. However, the treatment was greatly improved by lowering the pH from 9.5 to 6.5. | Removal of algae from Florida lakes by magnetic filtration. Magnetic filtration was used for the removal of algal populations present in five lakes located in the vicinity of Gainesville, Fla. It was found that the use of this technique enabled a good removal (94%) of algal cells from three lakes where the pH was around 7. The other two lakes, with a higher pH, displayed a lower removal. However, the treatment was greatly improved by lowering the pH from 9.5 to 6.5. | 2,106 |
pubmed23n0001_1105 | Factors influencing detection and enumeration of Pseudomonas aeruginosa by most-probable-number and membrane filtration techniques. | Most-probable-number (MPN) and membrane filtration (mF) techniques were evaluated with respect to selectivity, sensitivity, and efficiency in recovering Pseudomonas aeruginosa strains in hospital fluids and extramural water environments. Known numbers of cells of a naturally occurring strain of P. aeruginosa maintained in distilled water or cells subcultured on Standard Methods agar were added to test samples containing various types and levels of background microbial contamiants. Environmental samples containing unknown numbers of P. aeruginosa strains also were tested. Asparagine and acetamide broths were employed as presumptive media in MPN tests, and mPA and Pseudosel agars were used in mF assays. Statistical analyses of data showed the superiority and comparability of the asparagine-MPN and mPA-mF systems. Greater precision and accuracy were consistently obtained in either assay technique by the use of naturally occurring cells as test organisms. The type of filter and nature of diluents employed, as well as pH of assay media, were found to greatly influence both recovery and developemnt of characteristic colonial morphology in the mPA-mF system. | Factors influencing detection and enumeration of Pseudomonas aeruginosa by most-probable-number and membrane filtration techniques. Most-probable-number (MPN) and membrane filtration (mF) techniques were evaluated with respect to selectivity, sensitivity, and efficiency in recovering Pseudomonas aeruginosa strains in hospital fluids and extramural water environments. Known numbers of cells of a naturally occurring strain of P. aeruginosa maintained in distilled water or cells subcultured on Standard Methods agar were added to test samples containing various types and levels of background microbial contamiants. Environmental samples containing unknown numbers of P. aeruginosa strains also were tested. Asparagine and acetamide broths were employed as presumptive media in MPN tests, and mPA and Pseudosel agars were used in mF assays. Statistical analyses of data showed the superiority and comparability of the asparagine-MPN and mPA-mF systems. Greater precision and accuracy were consistently obtained in either assay technique by the use of naturally occurring cells as test organisms. The type of filter and nature of diluents employed, as well as pH of assay media, were found to greatly influence both recovery and developemnt of characteristic colonial morphology in the mPA-mF system. | 2,107 |
pubmed23n0001_1106 | Heat resistance of ileal loop reactive Bacillus cereus strains isolated from commercially canned food. | Sporeformers isolated from a commercially canned food were identified as Bacillus cereus, lactose-positive variants. The thermal resistance of spore crops produced from each of two representative cultures was determined in 0.067 M phosphate buffer at pH 7.0. The D121.1 values for one isolate were approximately 0.03 min (z = 9.9C), whereas the D121.1 values for the other isolate were 2.35 min (z = 7.9 C). Thermal inactivation results for heat-stressed isolates from each strain showed no significant alteration in heat resistance from that of the two parent spore crops. Both isolates were reactive when injected into the ligated rabbit ileum. | Heat resistance of ileal loop reactive Bacillus cereus strains isolated from commercially canned food. Sporeformers isolated from a commercially canned food were identified as Bacillus cereus, lactose-positive variants. The thermal resistance of spore crops produced from each of two representative cultures was determined in 0.067 M phosphate buffer at pH 7.0. The D121.1 values for one isolate were approximately 0.03 min (z = 9.9C), whereas the D121.1 values for the other isolate were 2.35 min (z = 7.9 C). Thermal inactivation results for heat-stressed isolates from each strain showed no significant alteration in heat resistance from that of the two parent spore crops. Both isolates were reactive when injected into the ligated rabbit ileum. | 2,108 |
pubmed23n0001_1107 | Kinetics of a bacterial culture growth: validity of the affinity rule in biological systems. | The kinetic study of a process is usually performed by measuring a convenient intensive property, P, as a function of time. The "affinity rule" states that, when a given process takes place under different external constraints (e.g., different temperatures, pressures, pH values, etc.), the various P versus time curves are related by an affinity transformation parallel to the time axis: in other words the P versus log time curves are parallel and can be superimposed by translation. The validity of the rule has been extensively tested in chemical and physiochemical processes, but there is no evidence as yet that it extends to biological systems. The present paper shows that the rule is indeed valid for the kinetics of growth of an Escherichia coli culture at various temperatures and pH values. More extended experiments are necessary to prove or disprove the general validity of the rule in biological systems, but its practical interest is evident: whenever it is valid it will be possible, from a very small number of measurements, to predict the complete behavior of the system in a number of various external conditions | Kinetics of a bacterial culture growth: validity of the affinity rule in biological systems. The kinetic study of a process is usually performed by measuring a convenient intensive property, P, as a function of time. The "affinity rule" states that, when a given process takes place under different external constraints (e.g., different temperatures, pressures, pH values, etc.), the various P versus time curves are related by an affinity transformation parallel to the time axis: in other words the P versus log time curves are parallel and can be superimposed by translation. The validity of the rule has been extensively tested in chemical and physiochemical processes, but there is no evidence as yet that it extends to biological systems. The present paper shows that the rule is indeed valid for the kinetics of growth of an Escherichia coli culture at various temperatures and pH values. More extended experiments are necessary to prove or disprove the general validity of the rule in biological systems, but its practical interest is evident: whenever it is valid it will be possible, from a very small number of measurements, to predict the complete behavior of the system in a number of various external conditions | 2,109 |
pubmed23n0001_1108 | Beclomethasone dipropionate aerosol in treatment of hay fever in children. | Eighteen children suffering from hay fever were treated with intra-nasal beclomethasone dipropionate (400 mug/day) and an identical placebo aerosol in a double-blind cross-over trial. 17 of the children preferred the intranasal beclomethasone dipropionate, one had no preference, none preferred the placebo. The effect on the nasal symptoms was impressive. Symptom scores decreased, on average, to 12% and the number of antihistamine tablets taken to 18% of the pretreatment amount. Some beneficial effect on eye symptoms was also discernible, possibly due to an indirect influence from the nasal mucosa via the nasolacrimal reflex. Adrenal function was not affected. It was concluded that 400 mug beclomethasone dipropionate given intranasally daily for some weeks is an effective and safe treatment for hay fever in children. | Beclomethasone dipropionate aerosol in treatment of hay fever in children. Eighteen children suffering from hay fever were treated with intra-nasal beclomethasone dipropionate (400 mug/day) and an identical placebo aerosol in a double-blind cross-over trial. 17 of the children preferred the intranasal beclomethasone dipropionate, one had no preference, none preferred the placebo. The effect on the nasal symptoms was impressive. Symptom scores decreased, on average, to 12% and the number of antihistamine tablets taken to 18% of the pretreatment amount. Some beneficial effect on eye symptoms was also discernible, possibly due to an indirect influence from the nasal mucosa via the nasolacrimal reflex. Adrenal function was not affected. It was concluded that 400 mug beclomethasone dipropionate given intranasally daily for some weeks is an effective and safe treatment for hay fever in children. | 2,110 |
pubmed23n0001_1109 | The influence of oxyhemoglobin affinity on tissue oxygen consumption. | In an intact animal or patient, any shift in oxyhemoglobin affinity is inevitably associated with concurrent fluctuation in numerous other determinants of oxygen delivery. For this reason, the influence of hemoglobin affinity for oxygen on tissue oxygen consumption has been incompletely evaluated. The purpose of this study was to investigate the influence of oxyhemoglobin affinity as the sole variable of oxygen delivery in an isolated perfused canine hindlimb. A membrane lung system which allowed precise control of blood flow, temperature, arterial oxygen content and arterial pH was established. Twelve isolated canine hindlimbs were alternatively perfused with autologous stored (left-shifted) and fresh (right-shifted) blood in paralled perfusion systems. The 2,3-DPG concentrations, P50 and oxygen consumptions were significantly different in the two paralled perfusion systems. A decreased hemoglobin affinity for oxygen appeared to permit increased oxygen off-loading at the tissue level. | The influence of oxyhemoglobin affinity on tissue oxygen consumption. In an intact animal or patient, any shift in oxyhemoglobin affinity is inevitably associated with concurrent fluctuation in numerous other determinants of oxygen delivery. For this reason, the influence of hemoglobin affinity for oxygen on tissue oxygen consumption has been incompletely evaluated. The purpose of this study was to investigate the influence of oxyhemoglobin affinity as the sole variable of oxygen delivery in an isolated perfused canine hindlimb. A membrane lung system which allowed precise control of blood flow, temperature, arterial oxygen content and arterial pH was established. Twelve isolated canine hindlimbs were alternatively perfused with autologous stored (left-shifted) and fresh (right-shifted) blood in paralled perfusion systems. The 2,3-DPG concentrations, P50 and oxygen consumptions were significantly different in the two paralled perfusion systems. A decreased hemoglobin affinity for oxygen appeared to permit increased oxygen off-loading at the tissue level. | 2,111 |
pubmed23n0001_1110 | The relationship between muscle surface pH and oxygen transport. | The relationship between muscle surface pH (pHM) and the arterial-venous oxygen content difference (AVO2D) was studied in 4 patients undergoing reconstructive arterial surgery and in 6 patients undergoing acute normovolemic hemodilution. There was a consistent inverse relation between pHM in the ischemic lower extremity and the femoral AVO2D before, during and after aortic clamping. There was also an inverse relation between AVO2D and pHM during hemodilution. These data confirm that pHM is a reliable indicator of tissue perfusion and correlates with the AVO2D. | The relationship between muscle surface pH and oxygen transport. The relationship between muscle surface pH (pHM) and the arterial-venous oxygen content difference (AVO2D) was studied in 4 patients undergoing reconstructive arterial surgery and in 6 patients undergoing acute normovolemic hemodilution. There was a consistent inverse relation between pHM in the ischemic lower extremity and the femoral AVO2D before, during and after aortic clamping. There was also an inverse relation between AVO2D and pHM during hemodilution. These data confirm that pHM is a reliable indicator of tissue perfusion and correlates with the AVO2D. | 2,112 |
pubmed23n0001_1111 | Sodium nitroprusside as a coronary vasodilator in man: a comparison of the effects of sodium nitroprusside and papaverine hydrochloride on aortocoronary saphenous vein graft flow. | Blood flow in aortocoronary saphenous vein grafts was studied in response to intragraft injection of sodium nitroprusside and papaverine hydrochloride. Following injection of 50 mug of sodium nitroprusside, mean graft flow increased from 40.1 +/- 4.5 to 81.3 +/- 8.5 ml per minute. Administration of 30 mg of papaverine hydrochloride caused mean graft flow to rise from 35.4 +/- 3.9 to 70 +/- 7.9 ml per minute. Sodium nitroprusside increases aortocoronary graft flow, the doubling effect of 50 mug of the drug being of the same order of magnitude as that induced by 30 mg of papaverine hydrochloride. | Sodium nitroprusside as a coronary vasodilator in man: a comparison of the effects of sodium nitroprusside and papaverine hydrochloride on aortocoronary saphenous vein graft flow. Blood flow in aortocoronary saphenous vein grafts was studied in response to intragraft injection of sodium nitroprusside and papaverine hydrochloride. Following injection of 50 mug of sodium nitroprusside, mean graft flow increased from 40.1 +/- 4.5 to 81.3 +/- 8.5 ml per minute. Administration of 30 mg of papaverine hydrochloride caused mean graft flow to rise from 35.4 +/- 3.9 to 70 +/- 7.9 ml per minute. Sodium nitroprusside increases aortocoronary graft flow, the doubling effect of 50 mug of the drug being of the same order of magnitude as that induced by 30 mg of papaverine hydrochloride. | 2,113 |
pubmed23n0001_1112 | Comparative evaluation of a new disposable rotating membrane oxygenator with bubble oxygenator. | The comparative in vivo performance of adult-size bubble and rotating membrane oxygenators was evaluated during closed-chest cardiopulmonary bypass for six hours in two groups of dogs. The results show that the rotating membrane oxygenator is efficient in oxygen and carbon dioxide transfer with minimal trauma to blood, while platelet destruction and hemolysis were marked with the bubble oxygenator. Cerebral, cardiac, and respiratory complications were frequent with the bubble oxygenator and absent with the membrane oxygenator. | Comparative evaluation of a new disposable rotating membrane oxygenator with bubble oxygenator. The comparative in vivo performance of adult-size bubble and rotating membrane oxygenators was evaluated during closed-chest cardiopulmonary bypass for six hours in two groups of dogs. The results show that the rotating membrane oxygenator is efficient in oxygen and carbon dioxide transfer with minimal trauma to blood, while platelet destruction and hemolysis were marked with the bubble oxygenator. Cerebral, cardiac, and respiratory complications were frequent with the bubble oxygenator and absent with the membrane oxygenator. | 2,114 |
pubmed23n0001_1113 | Adrenergic drug-receptor interaction in the presence of strontium (Sr++) in mammalian myocardium. | The specificity of Ca++ for the interaction of beta adrenergic agonists with their receptors in rabbit right atrial muscle was evaluated. This was accomplished by substituting Ca++ by an equimolar concentration of Sr++. Dose-response curves which demonstrate the effect of norepinephrine and isoproterenol on the rate of electrical activity in the presence of Ca++ or Sr++ were made. In addition, the antagonistic action of propranolol (1 X 10(-7) M) in a Ca++-containing or Sr++-containing medium was determined. The results clearly demonstrate that Sr++ can effectively substitute for Ca++ in maintaining electrical and mechanical activity in cardiac muscle. Also, norepinephrine and isoproterenol can increase the rate of electrical activity in a Ca++ or Sr++-containing medium. This effect of these beta agonists is mediated through the beta-receptors since propranolol effectively blocked their action. It appears that Ca++ per se is not required for beta agonist or antagonist-receptor interaction in cardiac muscle. The results are discussed in relation to the dependency on extracellular Ca++ for beta agonists to cause a change in the rate of electrical activity after receptor occupancy. | Adrenergic drug-receptor interaction in the presence of strontium (Sr++) in mammalian myocardium. The specificity of Ca++ for the interaction of beta adrenergic agonists with their receptors in rabbit right atrial muscle was evaluated. This was accomplished by substituting Ca++ by an equimolar concentration of Sr++. Dose-response curves which demonstrate the effect of norepinephrine and isoproterenol on the rate of electrical activity in the presence of Ca++ or Sr++ were made. In addition, the antagonistic action of propranolol (1 X 10(-7) M) in a Ca++-containing or Sr++-containing medium was determined. The results clearly demonstrate that Sr++ can effectively substitute for Ca++ in maintaining electrical and mechanical activity in cardiac muscle. Also, norepinephrine and isoproterenol can increase the rate of electrical activity in a Ca++ or Sr++-containing medium. This effect of these beta agonists is mediated through the beta-receptors since propranolol effectively blocked their action. It appears that Ca++ per se is not required for beta agonist or antagonist-receptor interaction in cardiac muscle. The results are discussed in relation to the dependency on extracellular Ca++ for beta agonists to cause a change in the rate of electrical activity after receptor occupancy. | 2,115 |
pubmed23n0001_1114 | Effects of isoprenaline and phenylephrine on plasma potassium: role of the liver. | A dog liver preparation in situ was used. Intravenous infusion of isoprenaline caused a decrease of plasma potassium levels, which was preceded, in some of the animals infused with higher doses, by a rise in plasma potassium. Propranolol abolished both these effects of isoprenaline, whereas phentolamine was devoided of effects. Liver potassium was not affected by isoprenaline infusions. Phenylephrine caused release of potassium from the liver; this effect was blocked by phentolamine, but not by propranolol. Combination of phenylephrine and isoprenaline induced a super-additive hyperkalemia. Analysis of these results led to the conclusion that the rise in plasma potassium due to phenylephrine might reflect a direct kalemotropic effect and an indirect hypoxemic effect. Isoprenaline seems to increase the hypoxemia caused by phenylephrine by opening the intrahepatic vascular shunts. | Effects of isoprenaline and phenylephrine on plasma potassium: role of the liver. A dog liver preparation in situ was used. Intravenous infusion of isoprenaline caused a decrease of plasma potassium levels, which was preceded, in some of the animals infused with higher doses, by a rise in plasma potassium. Propranolol abolished both these effects of isoprenaline, whereas phentolamine was devoided of effects. Liver potassium was not affected by isoprenaline infusions. Phenylephrine caused release of potassium from the liver; this effect was blocked by phentolamine, but not by propranolol. Combination of phenylephrine and isoprenaline induced a super-additive hyperkalemia. Analysis of these results led to the conclusion that the rise in plasma potassium due to phenylephrine might reflect a direct kalemotropic effect and an indirect hypoxemic effect. Isoprenaline seems to increase the hypoxemia caused by phenylephrine by opening the intrahepatic vascular shunts. | 2,116 |
pubmed23n0001_1115 | Benzodiazepines and amphetamine on avoidance behaviour in mice. | Six benzodiazepine derivatives, given alone or in combination with amphetamine, were tested in mice subjected to five 100-trial avoidance sessions in the shuttle-box. All derivatives, execpt bromazepam, showed some facilitating effects on avoidance responding when given alone. Facilitation was particularly evident following the administration of chlordiazepoxide (2.5 mg/kg), medazepam (10 mg/kg) and nitrazepam (0.25, 0.5 and 1 mg/kg). Favourable effects were obtained by combining each benzodiazepine compound with amphetamine. The levels of avoidance respinses were usually higher under benzodiazepine-amphetamine combinations than under benzodiazepines alone. | Benzodiazepines and amphetamine on avoidance behaviour in mice. Six benzodiazepine derivatives, given alone or in combination with amphetamine, were tested in mice subjected to five 100-trial avoidance sessions in the shuttle-box. All derivatives, execpt bromazepam, showed some facilitating effects on avoidance responding when given alone. Facilitation was particularly evident following the administration of chlordiazepoxide (2.5 mg/kg), medazepam (10 mg/kg) and nitrazepam (0.25, 0.5 and 1 mg/kg). Favourable effects were obtained by combining each benzodiazepine compound with amphetamine. The levels of avoidance respinses were usually higher under benzodiazepine-amphetamine combinations than under benzodiazepines alone. | 2,117 |
pubmed23n0001_1116 | [Age related effects of furosemide in the rat]. | Furosemide (6 mg/kg i.p.) increases the renal excretion of water, osmotic active substances, sodium and chloride in 5 to 33 day old rats more than in adults. The dose-response-relations are the same in rats of all age groups: 6 mg/kg of furosemide i.p. are very effective, an increase in dose to 30-60 mg/kg i.p. is not followed by a significantly higher efficacy. The increase in the renal excretion of potassium, hydrogen ions, ammonium and hydrogen carbonate by furosemide is also small in young rats. | [Age related effects of furosemide in the rat]. Furosemide (6 mg/kg i.p.) increases the renal excretion of water, osmotic active substances, sodium and chloride in 5 to 33 day old rats more than in adults. The dose-response-relations are the same in rats of all age groups: 6 mg/kg of furosemide i.p. are very effective, an increase in dose to 30-60 mg/kg i.p. is not followed by a significantly higher efficacy. The increase in the renal excretion of potassium, hydrogen ions, ammonium and hydrogen carbonate by furosemide is also small in young rats. | 2,118 |
pubmed23n0001_1117 | The effect of sulfhydryl reagents on the heart rate and coronary flow of the isolated perfused guinea-pig heart. | The action of a number of compounds able to react with thiols was tested on guinea-pig hearts perfused at constant pressure. The SH reagents used were NaNO2, oxidized glutathione, cystamine, diamide, 1,5-difluoro-2,4-dinitrobenzene, nitroglycerol, sodium nitroferricyanide and HgCl2. 6,6'-Dithiodinicotinic acid, an SH reagent that does not penetrate the cell, produced no effect. All the other SH reagents produced an increase in coronary flow. All except oxidized glutathione and nitroglycerol increased the heart rate. The increase in heart rate and oxygen consumption could be completely blocked by dichloroisoproterenol; the increase in coronary flow was not affected. Difluorodinitrobenzene, diamide, cystamine and NaNO2 significantly decreased the acid-soluble thiol content of the heart. For these compounds, there was a significant correlation between the decrease in coronary flow and the decrease in thiols. We conclude that in the isolated heart, most SH reagents, if used at the appropriate concentration, will increase the heart rate, probably by relaasing catecholamines. They will also decrease the coronary resistance, probably by a direct effect on the coronary vessels. | The effect of sulfhydryl reagents on the heart rate and coronary flow of the isolated perfused guinea-pig heart. The action of a number of compounds able to react with thiols was tested on guinea-pig hearts perfused at constant pressure. The SH reagents used were NaNO2, oxidized glutathione, cystamine, diamide, 1,5-difluoro-2,4-dinitrobenzene, nitroglycerol, sodium nitroferricyanide and HgCl2. 6,6'-Dithiodinicotinic acid, an SH reagent that does not penetrate the cell, produced no effect. All the other SH reagents produced an increase in coronary flow. All except oxidized glutathione and nitroglycerol increased the heart rate. The increase in heart rate and oxygen consumption could be completely blocked by dichloroisoproterenol; the increase in coronary flow was not affected. Difluorodinitrobenzene, diamide, cystamine and NaNO2 significantly decreased the acid-soluble thiol content of the heart. For these compounds, there was a significant correlation between the decrease in coronary flow and the decrease in thiols. We conclude that in the isolated heart, most SH reagents, if used at the appropriate concentration, will increase the heart rate, probably by relaasing catecholamines. They will also decrease the coronary resistance, probably by a direct effect on the coronary vessels. | 2,119 |
pubmed23n0001_1118 | Metabolic inhibition and adrenoceptor interconversion. | The adrenergic receptor responses of isolated strips of iris dilator muscle from rabbits were studied. An alpha agonist, norepinephrine and a beta agonist, isoprenaline, were used to assess adrenergic sensitivity before and after pretreatment of tissues with metabolic inhibitors at 22, 29 and 37 degrees C. The metabolic inhibitors used were iodoacetic acid and dinitrophenol. Temperature change altered adrenoceptor sensitivity in the same manner before and after metabolic inhibition. Iodoacetic acid (10.4 mug/ml) pretreatment increased both alpha and beta responses. Dinitrophenol (1.8 mug/ml) pretreatment increased alpha and decreased beta responsiveness. The results obtained indicate that some metabolic process altered by dinitrophenol may be involved in this adrenoceptor interconversion seen when temperature is changed. This supports the theory that local environment determines the drug sensitivity (alpha or beta) of a single adrenergic receptor. | Metabolic inhibition and adrenoceptor interconversion. The adrenergic receptor responses of isolated strips of iris dilator muscle from rabbits were studied. An alpha agonist, norepinephrine and a beta agonist, isoprenaline, were used to assess adrenergic sensitivity before and after pretreatment of tissues with metabolic inhibitors at 22, 29 and 37 degrees C. The metabolic inhibitors used were iodoacetic acid and dinitrophenol. Temperature change altered adrenoceptor sensitivity in the same manner before and after metabolic inhibition. Iodoacetic acid (10.4 mug/ml) pretreatment increased both alpha and beta responses. Dinitrophenol (1.8 mug/ml) pretreatment increased alpha and decreased beta responsiveness. The results obtained indicate that some metabolic process altered by dinitrophenol may be involved in this adrenoceptor interconversion seen when temperature is changed. This supports the theory that local environment determines the drug sensitivity (alpha or beta) of a single adrenergic receptor. | 2,120 |
pubmed23n0001_1119 | The influence of temperature increase, elevation of extracellular h+-concentration, and of triiodothyronine on the actions of phenylephrine, histamine, and beta-sympathomimetic drugs on rabbit aortic strips. | In the isolated preparation from the rabbit thoracic aorta, the affinities of the vasoconstrictor agents phenylephrine and histamine, as well as of the vasodilator beta-sympathomimetic drugs isoprenaline, fenoterol (TH 1165a), terbutaline, and salbutamol under the conditions of temperature increase, triiodothyronine and decrease of extracellular pH were investigated. It was observed that (1) a temperature increase from 25 degrees to 42 degrees C significantly indreased the maximal tension evoked by histamine, whereas that induced by the alpha-sympathomimetic drug phenylephrine was not altered significantly; the maximal relaxation caused by beta-sympathomimetic drugs either at 25 degrees or at 42 degrees C did not differ from one another; (2) the affinities of histamine, phenylephrine and of the beta-sympathomimetic drugs isoprenaline, fenoterol, terbutaline, and salbutamol each were comparable at either 25 degrees or 42 degrees C; the rank order of efficacy of the beta-sympathomimetic drugs is isoprenaline greater than fenoterol greater than salbutamol greater than terbutaline; (3) a decrease of the pH from 7.37 to 7.15 diminished the affinities of histamine and of the beta sympathomimetic drugs whereas that of the alpha-adrenergic drug phenylephrine was not altered. A further decrease of the pH to 6.8 diminished additionally the affinity of histamine and of isoprenaline, and especially that of the other beta-sympathomimetic drugs to such an extent that in the latter case complete dose-response curves could not be determined any more; (4) pretreatment of the animals with 0.4 mg/kg of triiodothyronine (T3) for two days, which strongly depressed the tension induced by either histamine or phenylephrine, did not alter the affinity of both drugs; T3 in vitro (10(-6) M) only diminished the affinity of histamine but left that of phenylephrine unaltered; pretreatment for two days with 0.2 mg/kg of T3 yielded a significant diminution of the pD2-values for two beta-sympathomimetic drugs investigated, namely isoprenaline and fenoterol; also the administration of T3 in vitro in a final concentration of 10(-6) M resulted in a diminution of the affinity of both beta-sympathomimetic drugs; (5) the results obtained show that also on the aorta beta-adrenoceptor stimulants are dependent on the metabolic state while alpha-adrenoceptor stimulants are not. | The influence of temperature increase, elevation of extracellular h+-concentration, and of triiodothyronine on the actions of phenylephrine, histamine, and beta-sympathomimetic drugs on rabbit aortic strips. In the isolated preparation from the rabbit thoracic aorta, the affinities of the vasoconstrictor agents phenylephrine and histamine, as well as of the vasodilator beta-sympathomimetic drugs isoprenaline, fenoterol (TH 1165a), terbutaline, and salbutamol under the conditions of temperature increase, triiodothyronine and decrease of extracellular pH were investigated. It was observed that (1) a temperature increase from 25 degrees to 42 degrees C significantly indreased the maximal tension evoked by histamine, whereas that induced by the alpha-sympathomimetic drug phenylephrine was not altered significantly; the maximal relaxation caused by beta-sympathomimetic drugs either at 25 degrees or at 42 degrees C did not differ from one another; (2) the affinities of histamine, phenylephrine and of the beta-sympathomimetic drugs isoprenaline, fenoterol, terbutaline, and salbutamol each were comparable at either 25 degrees or 42 degrees C; the rank order of efficacy of the beta-sympathomimetic drugs is isoprenaline greater than fenoterol greater than salbutamol greater than terbutaline; (3) a decrease of the pH from 7.37 to 7.15 diminished the affinities of histamine and of the beta sympathomimetic drugs whereas that of the alpha-adrenergic drug phenylephrine was not altered. A further decrease of the pH to 6.8 diminished additionally the affinity of histamine and of isoprenaline, and especially that of the other beta-sympathomimetic drugs to such an extent that in the latter case complete dose-response curves could not be determined any more; (4) pretreatment of the animals with 0.4 mg/kg of triiodothyronine (T3) for two days, which strongly depressed the tension induced by either histamine or phenylephrine, did not alter the affinity of both drugs; T3 in vitro (10(-6) M) only diminished the affinity of histamine but left that of phenylephrine unaltered; pretreatment for two days with 0.2 mg/kg of T3 yielded a significant diminution of the pD2-values for two beta-sympathomimetic drugs investigated, namely isoprenaline and fenoterol; also the administration of T3 in vitro in a final concentration of 10(-6) M resulted in a diminution of the affinity of both beta-sympathomimetic drugs; (5) the results obtained show that also on the aorta beta-adrenoceptor stimulants are dependent on the metabolic state while alpha-adrenoceptor stimulants are not. | 2,121 |
pubmed23n0001_1120 | Influence of pH on the contractor effect of convulsant barbiturate on frog lung. | Pentobarbial, thiopental and the convulsant 5-ethyl-5-(2-cyclohexylideneëthyl) barbituric acid (CHEB) were tested for contractor effect on the isolated lung of the fullfrog at pH 7.0 (7% CO2 and 20 mM HCO3-) and pH 8.4 (0.3% CO2 and 20mM HCO3-). CHEB was a potent contractor, thiopental a feeble contractor, and pentobarbital lacked contractor effect. The contractor potencies of both CHEB and thiopental were greater at the more acid pH. The potencies of formally charged agonists such as acetylcholine and K+ were not altered by the pH differences employed in these experiments. The pKa of CHEB was found to be 8.18 at 15 degrees C and 8.03 at 27 degrees C. Calculation of concentration-effect relationships of ionized and nonionized CHEB showed that only the nonionized CHEB was responsible for the contractor effect. | Influence of pH on the contractor effect of convulsant barbiturate on frog lung. Pentobarbial, thiopental and the convulsant 5-ethyl-5-(2-cyclohexylideneëthyl) barbituric acid (CHEB) were tested for contractor effect on the isolated lung of the fullfrog at pH 7.0 (7% CO2 and 20 mM HCO3-) and pH 8.4 (0.3% CO2 and 20mM HCO3-). CHEB was a potent contractor, thiopental a feeble contractor, and pentobarbital lacked contractor effect. The contractor potencies of both CHEB and thiopental were greater at the more acid pH. The potencies of formally charged agonists such as acetylcholine and K+ were not altered by the pH differences employed in these experiments. The pKa of CHEB was found to be 8.18 at 15 degrees C and 8.03 at 27 degrees C. Calculation of concentration-effect relationships of ionized and nonionized CHEB showed that only the nonionized CHEB was responsible for the contractor effect. | 2,122 |
pubmed23n0001_1121 | Action of six commonly used benzodiazepines on isolated guinea-pig ileum preparation. | The spasmolytic activity of six commonly used benzodiazepines was investigated on isolated guinea-pig ileum preparation. All six substances proved to be non-competitive antagonists of carbachol and barium chloride, the pD'2 values ranging between 3.23 and 4.37 in the presence of either agonist. The significance of these findings is discussed. | Action of six commonly used benzodiazepines on isolated guinea-pig ileum preparation. The spasmolytic activity of six commonly used benzodiazepines was investigated on isolated guinea-pig ileum preparation. All six substances proved to be non-competitive antagonists of carbachol and barium chloride, the pD'2 values ranging between 3.23 and 4.37 in the presence of either agonist. The significance of these findings is discussed. | 2,123 |
pubmed23n0001_1122 | Electrical events associated with the action of nicotine at the adrenergic nerve terminal. | Nicotine perfused through the coronary circulation of the isolated atropinized cat heart elicits antidromic activity in the cardiac sympathetic nerves. The pattern of discharge varies in a complex fashion with dose. At low concentrations, activity may last up to 10 min, whereas at high doses the antidromic response may last only a few seconds. Sympathetic transmitter is released into the perfusion fluid. There is dissociation between the amount of transmitter that overflows from the heart and the total antidromic activity with increasing dose of nicotine. With smaller doses of nicotine, the magnitude of the antidromic activity probably indicates the level of depolarization of the nerve terminal. Injection of greater doses of nicotine causes still greater transmitter release but not the generation of antidromic impulses, due presumably to persistent depolarization below a critical level. During the nicotine infusion and immediately afterwards, the antidromic response to acetylcholine and the effector responses associated with the adrenergic transmitter release by acetylcholine and sympathetic nerve stimulation were blocked. The rates of recovery of these responses were similar with a half-time of 4 to 5 min. Although the antidromic response to KCl was blocked during the nicotine infusion, it recovered more rapidly. Within 1-1.5 min, the antidromic response to KCl tended to exceed control levels. It is proposed that nicotine causes a prolonged depolarization of the membrane of the adrenergic nerve terminal. The site of action of nicotine, the basis of its prolonged action, and the interrelationship of this depolarization with transmitter release and intracellular uptake are discussed. | Electrical events associated with the action of nicotine at the adrenergic nerve terminal. Nicotine perfused through the coronary circulation of the isolated atropinized cat heart elicits antidromic activity in the cardiac sympathetic nerves. The pattern of discharge varies in a complex fashion with dose. At low concentrations, activity may last up to 10 min, whereas at high doses the antidromic response may last only a few seconds. Sympathetic transmitter is released into the perfusion fluid. There is dissociation between the amount of transmitter that overflows from the heart and the total antidromic activity with increasing dose of nicotine. With smaller doses of nicotine, the magnitude of the antidromic activity probably indicates the level of depolarization of the nerve terminal. Injection of greater doses of nicotine causes still greater transmitter release but not the generation of antidromic impulses, due presumably to persistent depolarization below a critical level. During the nicotine infusion and immediately afterwards, the antidromic response to acetylcholine and the effector responses associated with the adrenergic transmitter release by acetylcholine and sympathetic nerve stimulation were blocked. The rates of recovery of these responses were similar with a half-time of 4 to 5 min. Although the antidromic response to KCl was blocked during the nicotine infusion, it recovered more rapidly. Within 1-1.5 min, the antidromic response to KCl tended to exceed control levels. It is proposed that nicotine causes a prolonged depolarization of the membrane of the adrenergic nerve terminal. The site of action of nicotine, the basis of its prolonged action, and the interrelationship of this depolarization with transmitter release and intracellular uptake are discussed. | 2,124 |
pubmed23n0001_1123 | Positive inotropic effect of insulin on rabbit auricle in vitro. | Water soluble pig insulin (4 x 10(-8) to 4 x 10(-7) g/ml) produced a marked and long-lasting increase in the contractile force of the rabbit auricle in vitro. Once the maximum effect for a given insulin concentration had been reached, addition of more insulin did not produce any further increase in inotropic effect. Insulin was without effect in reserpinized animals. Inhibition of cardiac beta-receptors by propranolol suppressed the positive inotropic effect of insulin. These findings support the hypothesis that insulin releases catecholamines from the myocardium. | Positive inotropic effect of insulin on rabbit auricle in vitro. Water soluble pig insulin (4 x 10(-8) to 4 x 10(-7) g/ml) produced a marked and long-lasting increase in the contractile force of the rabbit auricle in vitro. Once the maximum effect for a given insulin concentration had been reached, addition of more insulin did not produce any further increase in inotropic effect. Insulin was without effect in reserpinized animals. Inhibition of cardiac beta-receptors by propranolol suppressed the positive inotropic effect of insulin. These findings support the hypothesis that insulin releases catecholamines from the myocardium. | 2,125 |
pubmed23n0001_1124 | Ionic basis for intracellular 14C-nicotine accumulation in slices from different rat brain areas. | The effects of ionic alterations on the accumulation, distribution and movements of 14C-nicotine in slices from rat brain striatum, hypothalamus, cortex and cerebellum were studied. The uptake of 14C-nicotone is not dependent upon Na+ present in the incubation fluid because a K+-substituted (O-Na+) solution increased the 14C-nicotine tissue space, a tris-substituted (O-Na+) solution decreased the 14C-nicotine tissue space and a sucrose-substituted (O-Na+) solution did not change the amount of 14C-nicotine taken up when compared to the 14C-nicotine tissue space obtained in a normal incubation solution. However, all three Na+-free solutions elicited a sustained decrease in 14C-nicotine efflux. The increase in 14C-nicotine space produced in a K+-substituted (O-Na+) solution was present primarily in the slower component of a two-component washout, whereas the decrease produced in a tris-substituted (O-Na+) solution produced an equal percentage decrease in the size of both components. Most of the observed effects could be attributed to a linear relationship between the logarithm of the intracellular K+ concentration and the 14C-nicotine tissue space. In conclusion, it appears that there is an intracellular binding site for nicotine and that the degree of binding is dependent upon the concentration of K+. | Ionic basis for intracellular 14C-nicotine accumulation in slices from different rat brain areas. The effects of ionic alterations on the accumulation, distribution and movements of 14C-nicotine in slices from rat brain striatum, hypothalamus, cortex and cerebellum were studied. The uptake of 14C-nicotone is not dependent upon Na+ present in the incubation fluid because a K+-substituted (O-Na+) solution increased the 14C-nicotine tissue space, a tris-substituted (O-Na+) solution decreased the 14C-nicotine tissue space and a sucrose-substituted (O-Na+) solution did not change the amount of 14C-nicotine taken up when compared to the 14C-nicotine tissue space obtained in a normal incubation solution. However, all three Na+-free solutions elicited a sustained decrease in 14C-nicotine efflux. The increase in 14C-nicotine space produced in a K+-substituted (O-Na+) solution was present primarily in the slower component of a two-component washout, whereas the decrease produced in a tris-substituted (O-Na+) solution produced an equal percentage decrease in the size of both components. Most of the observed effects could be attributed to a linear relationship between the logarithm of the intracellular K+ concentration and the 14C-nicotine tissue space. In conclusion, it appears that there is an intracellular binding site for nicotine and that the degree of binding is dependent upon the concentration of K+. | 2,126 |
pubmed23n0001_1125 | Mechanism of the antidiuretic effect of beta-adrenergic stimulation in man. | Beta-Adrenergic stimulation with isoproterenol hydrochloride in animals causes an antidiuresis similar to antidiuretic hormone. This investigation was undertaken to determine whether isoproterenol inhibits water diuresis in man. Seven young male volunteers were studied during water diuresis in three phases: (1) water-loading, (2) water-loading plus isoproterenol, and (3) water-loading plus isoproterenol plus propranolol hydrochloride. Antidiuresis occurred 20 minutes following isoproterenol infusion (0.03mug to 0.06mug/kg/min) from a mean of 19.4 to 2.0 ml/min. We found that antidiuresis is due to the hormonal (antidiuretic hormone) and nonhormonal changes (decreased glomerular filtration rate and renal plasma flow). These in turn are due to the cardiovascular effects of the drug. | Mechanism of the antidiuretic effect of beta-adrenergic stimulation in man. Beta-Adrenergic stimulation with isoproterenol hydrochloride in animals causes an antidiuresis similar to antidiuretic hormone. This investigation was undertaken to determine whether isoproterenol inhibits water diuresis in man. Seven young male volunteers were studied during water diuresis in three phases: (1) water-loading, (2) water-loading plus isoproterenol, and (3) water-loading plus isoproterenol plus propranolol hydrochloride. Antidiuresis occurred 20 minutes following isoproterenol infusion (0.03mug to 0.06mug/kg/min) from a mean of 19.4 to 2.0 ml/min. We found that antidiuresis is due to the hormonal (antidiuretic hormone) and nonhormonal changes (decreased glomerular filtration rate and renal plasma flow). These in turn are due to the cardiovascular effects of the drug. | 2,127 |
pubmed23n0001_1126 | Renal acidification in sickle cell trait. | Nine sickle cell trait and nine control subjects underwent six-hour ammonium chloride acid loading. Maximal urine osmolality and renal hemodynamics were studied separately. Base line arterial pH, carbon dioxide pressure (Pco2), and [HCO3] were normal and comparable in the two groups. After ammonium chloride loading, urine pH decreased to 5.3 or less in all, and maximal excretion of ammonium and titratable and net acid was comparable as was urine minus blood Pco2 after bicarbonate loading. The ammonium chloride acidosis caused a small decrease in red blood cell 2,3-diphosphoglycerate levels but no alteration in oxygen pressure at 50% saturation at pH 7.4, sickling, or adverse effects. Control and sickle cell trait subjects had comparable renal hemodynamics but maximal urine osmolality was lower in sickle-cell trait subjects. Adults with sickle cell trait have diminished renal concentrating ability and normal renal acidification and hemodynamics. | Renal acidification in sickle cell trait. Nine sickle cell trait and nine control subjects underwent six-hour ammonium chloride acid loading. Maximal urine osmolality and renal hemodynamics were studied separately. Base line arterial pH, carbon dioxide pressure (Pco2), and [HCO3] were normal and comparable in the two groups. After ammonium chloride loading, urine pH decreased to 5.3 or less in all, and maximal excretion of ammonium and titratable and net acid was comparable as was urine minus blood Pco2 after bicarbonate loading. The ammonium chloride acidosis caused a small decrease in red blood cell 2,3-diphosphoglycerate levels but no alteration in oxygen pressure at 50% saturation at pH 7.4, sickling, or adverse effects. Control and sickle cell trait subjects had comparable renal hemodynamics but maximal urine osmolality was lower in sickle-cell trait subjects. Adults with sickle cell trait have diminished renal concentrating ability and normal renal acidification and hemodynamics. | 2,128 |
pubmed23n0001_1127 | Therapeutic implications of gentamicin accumulation in severly diseased kidneys. | We evaluated the influence of severe disease in human kidneys (12 patients) on gentamicin sulfate accumulation characteristics in such tissue and compared the results with intrarenal tissue concentration data derived from the study of healthy dogs (54 kidneys) during variation in hydration and urinary pH. Our results indicate that, in the management of pyelonephritis complicating preexisting renal disease, if the minimal inhibitory gentamicin concentration for an infecting organism is greater than the usual therapeutic an nontoxic serum levels of the compound, then it may be appropriate to use alternate antibiotics that demonstrate lesser reduction in tissue drug accumulation in diseased kidneys. | Therapeutic implications of gentamicin accumulation in severly diseased kidneys. We evaluated the influence of severe disease in human kidneys (12 patients) on gentamicin sulfate accumulation characteristics in such tissue and compared the results with intrarenal tissue concentration data derived from the study of healthy dogs (54 kidneys) during variation in hydration and urinary pH. Our results indicate that, in the management of pyelonephritis complicating preexisting renal disease, if the minimal inhibitory gentamicin concentration for an infecting organism is greater than the usual therapeutic an nontoxic serum levels of the compound, then it may be appropriate to use alternate antibiotics that demonstrate lesser reduction in tissue drug accumulation in diseased kidneys. | 2,129 |
pubmed23n0001_1128 | The influence of pH on the growth and stability of transmissible gastroenteritis virus in vitro. | The influence of pH on the growth of transmissible gastroenteritis virus (TGEV) in adult pig thyroid cell culture, and on the stability of the virus was studied. At pH 7.2 and 100 fold higher than those at pH 8.0. The adsorption, penetration and uncoating steps of the viral replicative cycle were shown to be unaffected by pH variation. Synthesis of TGEV RNA during the first 12 hours post infection was found to be unaffected by pH variation between the range 6.5-8.0. After 12 hours breakdown of this RNA appeared to occur in cultures held at pH 7.2 and 8.0 but not at pH 6.5. When incubated at 37 degrees C for 24 hours the virus infectivity was found to be least affected by pH 6.5 but when kept at 4 degrees C for the same length of time, the virus infectivity remained constant between pH 5.0 and pH 8.0. | The influence of pH on the growth and stability of transmissible gastroenteritis virus in vitro. The influence of pH on the growth of transmissible gastroenteritis virus (TGEV) in adult pig thyroid cell culture, and on the stability of the virus was studied. At pH 7.2 and 100 fold higher than those at pH 8.0. The adsorption, penetration and uncoating steps of the viral replicative cycle were shown to be unaffected by pH variation. Synthesis of TGEV RNA during the first 12 hours post infection was found to be unaffected by pH variation between the range 6.5-8.0. After 12 hours breakdown of this RNA appeared to occur in cultures held at pH 7.2 and 8.0 but not at pH 6.5. When incubated at 37 degrees C for 24 hours the virus infectivity was found to be least affected by pH 6.5 but when kept at 4 degrees C for the same length of time, the virus infectivity remained constant between pH 5.0 and pH 8.0. | 2,142 |
pubmed23n0001_1129 | The influence of physicochemical factors on the thermal inactivation of murine interferon. | The degradation of biological activity of virus-induced murine interferon was determined in linear nonisothermal and multiple isothermal tests. The stabilizing effect of pH during heating on interferon in solution was greatest at low pH, such that pH 2 greater than pH 5 greater than pH 7 greater than or equal to pH 9; freeze-dried preparations of interferon were also more heat-stable at acid pH than at neutral pH. Heat stability was a function of the H+-ion concentration rather than the ionic composition of the buffer; interferon solutions containing monovalent cations with different ionic radii had similar heat stability. A change in the H+ ion concentration was a critical event during the cooling of heated interferon: a shift in the direction of acidity contributed to stability whereas a shift towards alkalinity led to inactivation. The rate of cooling of heated interferon significantly influenced its residual activity. Rapid cooling and sudden freezing decreased the residual activities of interferons at pH 2 and 9 more than "normal" cooling, an effect not observed at pH 7. Interferon heated to 80degree C could not be reactivated at 40degree C or 55degree C. Interferon of higher apparent molecular weight was more heat-stable than that with lower apparent molecular weight. It is postulated that the physicochemical alterations in the aqueous environment significantly affecting the stability of interferon operate by producing changes in the size and/or conformation of interferon molecules. A model is proposed that relates thermal inactivation to different possible molecular states of interferon. | The influence of physicochemical factors on the thermal inactivation of murine interferon. The degradation of biological activity of virus-induced murine interferon was determined in linear nonisothermal and multiple isothermal tests. The stabilizing effect of pH during heating on interferon in solution was greatest at low pH, such that pH 2 greater than pH 5 greater than pH 7 greater than or equal to pH 9; freeze-dried preparations of interferon were also more heat-stable at acid pH than at neutral pH. Heat stability was a function of the H+-ion concentration rather than the ionic composition of the buffer; interferon solutions containing monovalent cations with different ionic radii had similar heat stability. A change in the H+ ion concentration was a critical event during the cooling of heated interferon: a shift in the direction of acidity contributed to stability whereas a shift towards alkalinity led to inactivation. The rate of cooling of heated interferon significantly influenced its residual activity. Rapid cooling and sudden freezing decreased the residual activities of interferons at pH 2 and 9 more than "normal" cooling, an effect not observed at pH 7. Interferon heated to 80degree C could not be reactivated at 40degree C or 55degree C. Interferon of higher apparent molecular weight was more heat-stable than that with lower apparent molecular weight. It is postulated that the physicochemical alterations in the aqueous environment significantly affecting the stability of interferon operate by producing changes in the size and/or conformation of interferon molecules. A model is proposed that relates thermal inactivation to different possible molecular states of interferon. | 2,143 |
pubmed23n0001_1130 | Isoelectric focusing of herpes simplex virus. | Herpes simplex virus was inserted into a preformed stable pH gradient and electrofocused for about one hour. Dextran was used as the density gradient forming agent. Virions banded at pH 4.9+/-0.1 and nucleocapsids at 4.1+/-0.05. About 10-20 per cent of infective virus was recovered. | Isoelectric focusing of herpes simplex virus. Herpes simplex virus was inserted into a preformed stable pH gradient and electrofocused for about one hour. Dextran was used as the density gradient forming agent. Virions banded at pH 4.9+/-0.1 and nucleocapsids at 4.1+/-0.05. About 10-20 per cent of infective virus was recovered. | 2,144 |
pubmed23n0001_1131 | Effect of pH on the growth and cytopathogenicity of avian infectious bronchitis virus in chick kidney cells. | The growth of avian infectious bronchitis virus (IBV) in chick kidney cells at different pH values in the range 6.0-9.0 demonstrated that although the virus was released at a much faster rate at the higher pH values the titre tended to drop more quickly. At the acid pH values the virus was released more slowly but reached a maximum titre similar to that at the higher pH values and showed only minimum reduction in infectivity up to 49 hours post inoculation. The stability of virus in tissue culture medium was shown to be directly related to pH 6.0-8.0, being more stable at the acid pH values. The degree of cytopathogenicity induced in chick kidney cells following infection with IBV was directly related to the pH at which the cells were incubated, occurring earlier and more extensively in cells at the higher pH values. Cell macromolecule synthesis in chick kidney cells was inhibited following infection with IBV and was apparently due to cell damage and death. | Effect of pH on the growth and cytopathogenicity of avian infectious bronchitis virus in chick kidney cells. The growth of avian infectious bronchitis virus (IBV) in chick kidney cells at different pH values in the range 6.0-9.0 demonstrated that although the virus was released at a much faster rate at the higher pH values the titre tended to drop more quickly. At the acid pH values the virus was released more slowly but reached a maximum titre similar to that at the higher pH values and showed only minimum reduction in infectivity up to 49 hours post inoculation. The stability of virus in tissue culture medium was shown to be directly related to pH 6.0-8.0, being more stable at the acid pH values. The degree of cytopathogenicity induced in chick kidney cells following infection with IBV was directly related to the pH at which the cells were incubated, occurring earlier and more extensively in cells at the higher pH values. Cell macromolecule synthesis in chick kidney cells was inhibited following infection with IBV and was apparently due to cell damage and death. | 2,145 |
pubmed23n0001_1132 | Eugenol improvement due to aging. A laboratory appraisal. | The characteristics of eugenol for dental use appear to improve with age. Exposure to air and light causes certain chemical and physical changes, but the exact nature of the age improvement requires further elucidation. | Eugenol improvement due to aging. A laboratory appraisal. The characteristics of eugenol for dental use appear to improve with age. Exposure to air and light causes certain chemical and physical changes, but the exact nature of the age improvement requires further elucidation. | 2,147 |
pubmed23n0001_1133 | Effect of prednisolone and salicyclic acid on ionic fluxes across the human stomach. | We compared ionic fluxes across human gastric mucosa after instillation of test solutions of isotonic hydrochloric acid alone or containing salicylic acid, or prednisolone or both. Prednisolone produced no alteration in fluxes of H+ and Na+ ions compared with controls. Salicyclic acid induced a significant net loss of H+ ions and gain of Na+ ions indicating alteration of the gastric mucosal barrier. Combined salicyclic acid plus prednisolone produced no increase in permeability of gastric mucosa to H+ and Na+ ions or to salicylic acid itself. Prednisolone was not appreciably absorbed from the stomach while salicylic acid was well absorbed. Combination of salicylic acid and prednisolone did not increase absorption of either drug. Neither salicylic acid nor prednisolone solutions alone or combined caused an increase in pepsin output over that due to 160 mM HCl. Salicylic acid resulted in a significant fall in potential difference compared to control while prednisolone produced no change in the one subject studied. In acute studies in man prednisolone is not absorbed from the stomach, does not itself affect the gastric mucosal barrier nor pepsin output nor does it enhance the absorption of or effect of salicylic acid on gastric ionic fluxes or pepsin output. | Effect of prednisolone and salicyclic acid on ionic fluxes across the human stomach. We compared ionic fluxes across human gastric mucosa after instillation of test solutions of isotonic hydrochloric acid alone or containing salicylic acid, or prednisolone or both. Prednisolone produced no alteration in fluxes of H+ and Na+ ions compared with controls. Salicyclic acid induced a significant net loss of H+ ions and gain of Na+ ions indicating alteration of the gastric mucosal barrier. Combined salicyclic acid plus prednisolone produced no increase in permeability of gastric mucosa to H+ and Na+ ions or to salicylic acid itself. Prednisolone was not appreciably absorbed from the stomach while salicylic acid was well absorbed. Combination of salicylic acid and prednisolone did not increase absorption of either drug. Neither salicylic acid nor prednisolone solutions alone or combined caused an increase in pepsin output over that due to 160 mM HCl. Salicylic acid resulted in a significant fall in potential difference compared to control while prednisolone produced no change in the one subject studied. In acute studies in man prednisolone is not absorbed from the stomach, does not itself affect the gastric mucosal barrier nor pepsin output nor does it enhance the absorption of or effect of salicylic acid on gastric ionic fluxes or pepsin output. | 2,149 |
pubmed23n0001_1134 | Effects of altering monoamine metabolism on the adrenocortical response to hypoxia. | Anesthetized dogs, which had been prepared with lumboadrenal vein cannulae, were intravenously infused with monoamine axidase (alphaETA), tryptophan hydroxylase (pCPA) or tyrosine hydroxylase (alphaMT) inhibitors 30 min prior to exposure to 10% oxygen at ground level. These studies were designed to ascertain the role of the neurotransmitters, serotonin and norepinephrine, in the adrenocortical response to hypoxia. In normoxic animals, alphaETA decreased basal cortisol secretion and increased systolic pressure, whereas pCPA and alphaMT were essentially without afffect on these parameters. All inhibitors prevented the rise in cortisol secretion usually observed in hypoxic dogs. Alpha ETA appeared to inhibit the adrenocortical response to hypoxia as a result of its potent pressore activity, while pCPA and alphaMT inhibited cortisol secretion by interfering with the synthesis of serotonin and norepinephrine, respictively. These data suggest that substances which alter the content and/or turnover of brain monoamines abolish the hypoxic rise in cortisol secretion and thus would lower the resistance of the animal to this stressor. | Effects of altering monoamine metabolism on the adrenocortical response to hypoxia. Anesthetized dogs, which had been prepared with lumboadrenal vein cannulae, were intravenously infused with monoamine axidase (alphaETA), tryptophan hydroxylase (pCPA) or tyrosine hydroxylase (alphaMT) inhibitors 30 min prior to exposure to 10% oxygen at ground level. These studies were designed to ascertain the role of the neurotransmitters, serotonin and norepinephrine, in the adrenocortical response to hypoxia. In normoxic animals, alphaETA decreased basal cortisol secretion and increased systolic pressure, whereas pCPA and alphaMT were essentially without afffect on these parameters. All inhibitors prevented the rise in cortisol secretion usually observed in hypoxic dogs. Alpha ETA appeared to inhibit the adrenocortical response to hypoxia as a result of its potent pressore activity, while pCPA and alphaMT inhibited cortisol secretion by interfering with the synthesis of serotonin and norepinephrine, respictively. These data suggest that substances which alter the content and/or turnover of brain monoamines abolish the hypoxic rise in cortisol secretion and thus would lower the resistance of the animal to this stressor. | 2,152 |
pubmed23n0001_1135 | Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes. | 1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors. | Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes. 1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors. | 2,153 |
pubmed23n0001_1136 | Stabilization of rat liver tyrosine aminotransferase by tetracycline. | Rat liver tyrosine aminotransferase was purified 200-fold and an antiserum raised against it in rabbits. 2. Hepatic tyrosine aminotransferase activity was increased fourfold by tyrosine, twofold by tetracycline, 2.5-fold by cortisone 21-acetate and ninefold by a combination of tyrosine and cortisol administered intraperitoneally to rats. 3. Radioimmunoassay with 14C-labelled tyrosine aminotransferase, in conjunction with rabbit antiserum against the enzyme, revealed that cortisol stimulates the synthesis of the enzyme de novo, but that tetracycline has no such effect. 4. Incubation of rat liver homogenates with purified tyrosine aminotransferase in vitro leads to a rapid inactivation of the enzyme, which tetracycline partially inhibits. 5. The inactivation is brought about by intact lysosomes, and the addition of 10mM-cysteine increases the rate of enzyme inactivation, which is further markedly increased by 10mM-Mg2+ and 10mM-ATP. Here again tetracycline partially inhibits the decay rate, leading to the inference that the increase of tyrosine aminotransferase activity in vivo by tetracycline is brought about by the latter inhibiting the lysosomal catheptic action. | Stabilization of rat liver tyrosine aminotransferase by tetracycline. Rat liver tyrosine aminotransferase was purified 200-fold and an antiserum raised against it in rabbits. 2. Hepatic tyrosine aminotransferase activity was increased fourfold by tyrosine, twofold by tetracycline, 2.5-fold by cortisone 21-acetate and ninefold by a combination of tyrosine and cortisol administered intraperitoneally to rats. 3. Radioimmunoassay with 14C-labelled tyrosine aminotransferase, in conjunction with rabbit antiserum against the enzyme, revealed that cortisol stimulates the synthesis of the enzyme de novo, but that tetracycline has no such effect. 4. Incubation of rat liver homogenates with purified tyrosine aminotransferase in vitro leads to a rapid inactivation of the enzyme, which tetracycline partially inhibits. 5. The inactivation is brought about by intact lysosomes, and the addition of 10mM-cysteine increases the rate of enzyme inactivation, which is further markedly increased by 10mM-Mg2+ and 10mM-ATP. Here again tetracycline partially inhibits the decay rate, leading to the inference that the increase of tyrosine aminotransferase activity in vivo by tetracycline is brought about by the latter inhibiting the lysosomal catheptic action. | 2,154 |
pubmed23n0001_1137 | Factors regulating amino acid release from extrasplanchnic tissues in the rat. Interactions of alanine and glutamine. | 1. Factors regulating the release of alanine and glutamine in vivo were investigated in starved rats by removing the liver from the circulation and monitoring blood metabolite changes for 30 min. 2. Alanine and glutamine were the predominant amino acids released into the circulation in this preparation. 3. Dichloroacetate, an activator of pyruvate dehydrogenase, inhibited net alanine release: it also interfered with the metabolism of the branched-chain amino acids valine, leucine and isoleucine. 4. L-Cycloserine, an inhibitor of alanine aminotransferase, decreased alanine accumulation by 80% after functional hepatectomy, whereas methionine sulphoximine, an inhibitor of glutamine synthetase, decreased glutamine accumulation by the same amount. 5. It was concluded that: (a) the alanine aminotransferase and the glutamine synthetase pathways respectively were responsible for 80% of the alanine and glutamine released into the circulation by the extrasplanchnic tissues, and extrahepatic proteolysis could account for a maximum of 20%; (b) alanine formation by the peripheral tissues was dependent on availability of pyruvate and not of glutamate; (c) glutamate availability could influence glutamine formation subject, possibly, to renal control. | Factors regulating amino acid release from extrasplanchnic tissues in the rat. Interactions of alanine and glutamine. 1. Factors regulating the release of alanine and glutamine in vivo were investigated in starved rats by removing the liver from the circulation and monitoring blood metabolite changes for 30 min. 2. Alanine and glutamine were the predominant amino acids released into the circulation in this preparation. 3. Dichloroacetate, an activator of pyruvate dehydrogenase, inhibited net alanine release: it also interfered with the metabolism of the branched-chain amino acids valine, leucine and isoleucine. 4. L-Cycloserine, an inhibitor of alanine aminotransferase, decreased alanine accumulation by 80% after functional hepatectomy, whereas methionine sulphoximine, an inhibitor of glutamine synthetase, decreased glutamine accumulation by the same amount. 5. It was concluded that: (a) the alanine aminotransferase and the glutamine synthetase pathways respectively were responsible for 80% of the alanine and glutamine released into the circulation by the extrasplanchnic tissues, and extrahepatic proteolysis could account for a maximum of 20%; (b) alanine formation by the peripheral tissues was dependent on availability of pyruvate and not of glutamate; (c) glutamate availability could influence glutamine formation subject, possibly, to renal control. | 2,155 |
pubmed23n0001_1138 | Amino acids attached to transfer ribonucleic acid in vivo. | 1. tRNA was extracted from rabbit liver by both the phenol and diethyl pyrocarbonate methods under conditions preventing deacylation of the amino acids attached in vivo. 2. After deacylation 12 amino acids were determined by gas-liquid chromatography, by using the flame-ionization and nitrogen-sensitive thermionic detectors. 3. Comparison of the distribution of 12 amino acids attached to tRNA with those contained in total tissue protein and in the free pool showed little correlation. 4. Results for the enzymic charging assay for tRNA in vitro did not correlate satisfactorily with the analysis of amino acids attached to tRNA in vivo. Marked differences were ntoed in comparison made between our own and other published results. | Amino acids attached to transfer ribonucleic acid in vivo. 1. tRNA was extracted from rabbit liver by both the phenol and diethyl pyrocarbonate methods under conditions preventing deacylation of the amino acids attached in vivo. 2. After deacylation 12 amino acids were determined by gas-liquid chromatography, by using the flame-ionization and nitrogen-sensitive thermionic detectors. 3. Comparison of the distribution of 12 amino acids attached to tRNA with those contained in total tissue protein and in the free pool showed little correlation. 4. Results for the enzymic charging assay for tRNA in vitro did not correlate satisfactorily with the analysis of amino acids attached to tRNA in vivo. Marked differences were ntoed in comparison made between our own and other published results. | 2,156 |
pubmed23n0001_1139 | Initial rates of pyruvate transport in mitochondria determined by an "inhibitor-stop" technique. | An "inhibitor-stop" technique has been developed for measuring initial rates of pyruvate transport into mitochondria. The technique uses alpha-cyano-3-hydroxycinnamate as the inhibitor and separates the mitochondria from the radioactive medium by Millipore filtration. Observed rates depend on availability of hydroxyl and other exchangeable anions within the mitochondrial matrix. | Initial rates of pyruvate transport in mitochondria determined by an "inhibitor-stop" technique. An "inhibitor-stop" technique has been developed for measuring initial rates of pyruvate transport into mitochondria. The technique uses alpha-cyano-3-hydroxycinnamate as the inhibitor and separates the mitochondria from the radioactive medium by Millipore filtration. Observed rates depend on availability of hydroxyl and other exchangeable anions within the mitochondrial matrix. | 2,157 |
pubmed23n0001_1140 | A comparative study between a chondroitinase B and a chondroitinase AC from Flavobacterium heparinum: Isolation of a chondroitinase AC-susceptible dodecasaccharide from chondroitin sulphate B. | A chondroitinase that degrades only chondroitin sulphate B was isolated from Flavobacterium heparinum, and separated from a constitutive chondroitinase AC also present in extracts of F. heparinum. The enzyme acts only on chondroitin sulphate B, producing oligo- and tetra-saccharides, plus an unsaturated 4-sulphated disaccharide (deltaDi-4S). The oligosaccharide fraction (mol. wt. 3000) is susceptible to chondroitinase AC, producing mainly deltaDi-4S. The chondroitinase B is distinguished from chondroitinase AC by several properties, such as the effect of certain metal ions, temperature for optimal activity, and susceptibility to increasing salt concentrations. The enzyme is induced in F. heparinum by all the chondroitin sulphates, as well as by the disaccharides prepared from the chondroitins. The mechanism of induction of the enzyme and the structure of chondroitin sulphate B are discussed in relation to these results. | A comparative study between a chondroitinase B and a chondroitinase AC from Flavobacterium heparinum: Isolation of a chondroitinase AC-susceptible dodecasaccharide from chondroitin sulphate B. A chondroitinase that degrades only chondroitin sulphate B was isolated from Flavobacterium heparinum, and separated from a constitutive chondroitinase AC also present in extracts of F. heparinum. The enzyme acts only on chondroitin sulphate B, producing oligo- and tetra-saccharides, plus an unsaturated 4-sulphated disaccharide (deltaDi-4S). The oligosaccharide fraction (mol. wt. 3000) is susceptible to chondroitinase AC, producing mainly deltaDi-4S. The chondroitinase B is distinguished from chondroitinase AC by several properties, such as the effect of certain metal ions, temperature for optimal activity, and susceptibility to increasing salt concentrations. The enzyme is induced in F. heparinum by all the chondroitin sulphates, as well as by the disaccharides prepared from the chondroitins. The mechanism of induction of the enzyme and the structure of chondroitin sulphate B are discussed in relation to these results. | 2,158 |
pubmed23n0001_1141 | Subtilopeptidase A isoenzyme system. Interaction with serum components and its importance for quantitative immunoelectrophoresis. | A method was developed which involved electroimmunoassay and crossed immunoelectrophoresis of subtilopeptidase A (EC 3.4.21.14). Initial trials with unfractionated antiserum were not successful and interaction of the enzyme with non-immunoglobulin serum components were shown to be the cause of the failures. Quantitative immunoelectrophoresis was possible when purified immunoglobulins were used. A pH of 6.5 (lower than the usual pH 8.6) was necessary to obtain a proper baseline definition. Subtilopeptidase A was confirmed as a multiple isoenzyme system. Qualitative inter-batch variations were detected. Di-isopropyl phosphorofluoridate inhibition altered the electrophoretic pattern, but no loss of antigenic determinants was observed. | Subtilopeptidase A isoenzyme system. Interaction with serum components and its importance for quantitative immunoelectrophoresis. A method was developed which involved electroimmunoassay and crossed immunoelectrophoresis of subtilopeptidase A (EC 3.4.21.14). Initial trials with unfractionated antiserum were not successful and interaction of the enzyme with non-immunoglobulin serum components were shown to be the cause of the failures. Quantitative immunoelectrophoresis was possible when purified immunoglobulins were used. A pH of 6.5 (lower than the usual pH 8.6) was necessary to obtain a proper baseline definition. Subtilopeptidase A was confirmed as a multiple isoenzyme system. Qualitative inter-batch variations were detected. Di-isopropyl phosphorofluoridate inhibition altered the electrophoretic pattern, but no loss of antigenic determinants was observed. | 2,159 |
pubmed23n0001_1142 | Fructose 1,6-diphosphate aldolase from rabbit muscle. Effect of pH on the rate of formation and on the equilibrium concentration of the carbanion intermediate. | The rate of oxidation of ferricyanide of the aldolase-dihydroxyacetone phosphate complex was measured under different conditions. The following conclusions are drawn. 1. In the cleavage of fructose diphosphate, catalysed by native aldolase, the steady-state concentration of the enzyme-dihydroxyacetone phosphate carbanion intermediate represents less than 6% of the total enzyme-substrate intermediates. 2. Fructose diphosphate and dihydroxyacetone phosphate compete for the four catalytic sites on aldolase, the binding of fructose diphosphate being about twice as tight. 3. The equilibrium concentration of the carbanion intermediate formed by reaction of carboxypeptidase-treated aldolase with dihydroxyacetone phosphate is independent of pH between 5.0 and 9.0. The rates of fromation of the carbanion intermediate and of the reverse reaction are, however, concomitantly increased by increasing pH between 5.0 and 6.5. | Fructose 1,6-diphosphate aldolase from rabbit muscle. Effect of pH on the rate of formation and on the equilibrium concentration of the carbanion intermediate. The rate of oxidation of ferricyanide of the aldolase-dihydroxyacetone phosphate complex was measured under different conditions. The following conclusions are drawn. 1. In the cleavage of fructose diphosphate, catalysed by native aldolase, the steady-state concentration of the enzyme-dihydroxyacetone phosphate carbanion intermediate represents less than 6% of the total enzyme-substrate intermediates. 2. Fructose diphosphate and dihydroxyacetone phosphate compete for the four catalytic sites on aldolase, the binding of fructose diphosphate being about twice as tight. 3. The equilibrium concentration of the carbanion intermediate formed by reaction of carboxypeptidase-treated aldolase with dihydroxyacetone phosphate is independent of pH between 5.0 and 9.0. The rates of fromation of the carbanion intermediate and of the reverse reaction are, however, concomitantly increased by increasing pH between 5.0 and 6.5. | 2,160 |
pubmed23n0001_1143 | Purification and characterization of a collagenase extracted from rabbit tumours. | A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of alpha 1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as mumol of collagen in solution cleaved/h per mg of enzyme at 35 degrees C, WAS 1.74.2. The collagenase had a broad pH optimum from pH7.0 to 9.5, and a mol.wt. of between 33000 and 35000. It was inhibited by dithiothreitol, L-cysteine, D-penicillamine, EDTA and 1,10-phenanthroline, and by both rabbit and human serum. 3. Removal of cations by a chelating resin (Chelex 100) produced as inactive enzyme that could be reactiviated by the addition of Ca2+ ions at concentrations as low as 1muM. Other bivalent cations were not effective. 4. The purified collagenase cleaved peptides alpha2 and alpha1-CB7 (denatured polypeptides of collagen) at 37 degrees C at one site only. [alpha1 (I)]2alpha2 and [alpha1(III)]3 collagens in solution were cleaved at the same site approximately five times more rapidly than [alpha1 (II)]3. 5. An inhibitor of the enzyme in the tumour extracts, which was dissociable from the enzyme at the (NH4) 2SO4 precipitation step of purification, had a mol. wt. of between 40000 and 50000 but was distinct from the alpha1 trypsin inhibitor. 6. Studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes. | Purification and characterization of a collagenase extracted from rabbit tumours. A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of alpha 1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as mumol of collagen in solution cleaved/h per mg of enzyme at 35 degrees C, WAS 1.74.2. The collagenase had a broad pH optimum from pH7.0 to 9.5, and a mol.wt. of between 33000 and 35000. It was inhibited by dithiothreitol, L-cysteine, D-penicillamine, EDTA and 1,10-phenanthroline, and by both rabbit and human serum. 3. Removal of cations by a chelating resin (Chelex 100) produced as inactive enzyme that could be reactiviated by the addition of Ca2+ ions at concentrations as low as 1muM. Other bivalent cations were not effective. 4. The purified collagenase cleaved peptides alpha2 and alpha1-CB7 (denatured polypeptides of collagen) at 37 degrees C at one site only. [alpha1 (I)]2alpha2 and [alpha1(III)]3 collagens in solution were cleaved at the same site approximately five times more rapidly than [alpha1 (II)]3. 5. An inhibitor of the enzyme in the tumour extracts, which was dissociable from the enzyme at the (NH4) 2SO4 precipitation step of purification, had a mol. wt. of between 40000 and 50000 but was distinct from the alpha1 trypsin inhibitor. 6. Studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes. | 2,161 |
pubmed23n0001_1144 | Inhibition of leucocytic lysosomal enzymes by glycosaminoglycans in vitro. | 1. A lysosomal fraction was separated by density-gradient centrifugation from a highly purified human polymorphonuclear leucocyte suspension. 2. Some 23 different lysosomal enzymes were assayed for activity in the presence of various concentrations of glycosaminoglycans. 3. The 21 acid hydrolases assayed were strongly inhibited to different degrees by low (0-12 mmol/l) concentrations of glycosaminoglycans in a pH-dependent manner. Thus inhibitions were stronger below pH4.5, with activity returning to control values at about pH5.0. 4. On a molar basis, the inhibitory activity for the several glycosaminoglycans studied was: heparin greater than chondroitin sulphate greater than hyaluronic acid. 5. Once the glycosaminoglycan-acid hydrolase complex was formed, it was partially dissociated by slight elevations in the pH of the incubation medium, by increasing the ionic strength of the incubation medium, or by adding several cationic proteins (e.g. histone, protamine). 6. As leucocytic lysosomes contain large amounts of chondroitin sulphate, and have a strongly acid intragranular pH, we suggest that glycosaminoglycans may modify lysosomal function through the formation of complexes with lysosomal enzymes, by inhibiting the digestive activity of the acid hydrolases when the intralysosomal pH is below their pI. | Inhibition of leucocytic lysosomal enzymes by glycosaminoglycans in vitro. 1. A lysosomal fraction was separated by density-gradient centrifugation from a highly purified human polymorphonuclear leucocyte suspension. 2. Some 23 different lysosomal enzymes were assayed for activity in the presence of various concentrations of glycosaminoglycans. 3. The 21 acid hydrolases assayed were strongly inhibited to different degrees by low (0-12 mmol/l) concentrations of glycosaminoglycans in a pH-dependent manner. Thus inhibitions were stronger below pH4.5, with activity returning to control values at about pH5.0. 4. On a molar basis, the inhibitory activity for the several glycosaminoglycans studied was: heparin greater than chondroitin sulphate greater than hyaluronic acid. 5. Once the glycosaminoglycan-acid hydrolase complex was formed, it was partially dissociated by slight elevations in the pH of the incubation medium, by increasing the ionic strength of the incubation medium, or by adding several cationic proteins (e.g. histone, protamine). 6. As leucocytic lysosomes contain large amounts of chondroitin sulphate, and have a strongly acid intragranular pH, we suggest that glycosaminoglycans may modify lysosomal function through the formation of complexes with lysosomal enzymes, by inhibiting the digestive activity of the acid hydrolases when the intralysosomal pH is below their pI. | 2,162 |
pubmed23n0001_1145 | Circulating levels of prolactin in human breast cancer. | Serum prolactin concentrations were measured by radioimmunoassays in 98 patients with established carcinoma of breast, 12 patients with cystic mastitis and 10 patients with gynaecomastia and compared with that of age matched normal control women. The serum prolactin levels in the patients with breast cancer, gynaecomastia or cystic mastitis were observed to be similar to that in normal women. It was interesting to note that the levels of prolactin in the luteal phase of the cycle were higher than that in the early follicular phase in normal women. | Circulating levels of prolactin in human breast cancer. Serum prolactin concentrations were measured by radioimmunoassays in 98 patients with established carcinoma of breast, 12 patients with cystic mastitis and 10 patients with gynaecomastia and compared with that of age matched normal control women. The serum prolactin levels in the patients with breast cancer, gynaecomastia or cystic mastitis were observed to be similar to that in normal women. It was interesting to note that the levels of prolactin in the luteal phase of the cycle were higher than that in the early follicular phase in normal women. | 2,274 |
pubmed23n0001_1146 | Effect of aflatoxin B1 on pyridine nucleotides and NADP linked dehydrogenases. | The effect of a single interaperitoneal injection (6 mg/kg body weight) of aflatoxin B1 in propylene glycol on pyridine nucleotides and NDP linked dehydrogenases was studied 24 h after administration of the toxin. The liver showed a decrease in total proteins and pyridine nucleotides though levels of NADP and NADPH remained unchanged. Levels of NAD and NADH were decreased. The activities of hepatic of hwpRIX of hepatic malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) were not altered though ICDH showed an increase when expressed on protein basis. However, there was a significance decrease in the activity of combined HMP dehydrogenases. Adipose tissue showed increased activities of the HMP dehydrogenasess. | Effect of aflatoxin B1 on pyridine nucleotides and NADP linked dehydrogenases. The effect of a single interaperitoneal injection (6 mg/kg body weight) of aflatoxin B1 in propylene glycol on pyridine nucleotides and NDP linked dehydrogenases was studied 24 h after administration of the toxin. The liver showed a decrease in total proteins and pyridine nucleotides though levels of NADP and NADPH remained unchanged. Levels of NAD and NADH were decreased. The activities of hepatic of hwpRIX of hepatic malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) were not altered though ICDH showed an increase when expressed on protein basis. However, there was a significance decrease in the activity of combined HMP dehydrogenases. Adipose tissue showed increased activities of the HMP dehydrogenasess. | 2,275 |
pubmed23n0001_1147 | Esterase activity of zinc neutral proteases. | The hydrolysis of a series of depsipeptides demonstrates that the zinc neutral endopeptidases of bacteria are active esterases. Esters such as BzGly-OPhe-Ala, BzGly-OLeu-Ala, and FA-Gly-OLeu-NH2 are hydrolyzed at rates three- to eightfold slower than are their exact peptide analogues, when hydrolyzed by thermolysin, Bacillus subtilis neutral protease and the neutral protease from Aeromonas proteolytica. Ester hydrolysis by zinc neutral proteases follows the characteristic preference for hydrophobic amino acids adjacent to the site of cleavage, discerned from the hydrolysis of peptide substrates. Removal of zinc from thermolysin abolishes the esterase activity of the native enzyme. Among the metals examined, only Co2+ and Zn2+ restore esterase activity to any significant extent, Co2+ restoring 50% and Zn2+ 100% of the native thermolysin activity. The hydrolysis of esters and peptides by thermolysin does not differ with respect to either the binding or catalytic steps. Substrate specificity, pH-rate profiles, inhibitor, and deuterium isotope effects are identical for both types of substrates. | Esterase activity of zinc neutral proteases. The hydrolysis of a series of depsipeptides demonstrates that the zinc neutral endopeptidases of bacteria are active esterases. Esters such as BzGly-OPhe-Ala, BzGly-OLeu-Ala, and FA-Gly-OLeu-NH2 are hydrolyzed at rates three- to eightfold slower than are their exact peptide analogues, when hydrolyzed by thermolysin, Bacillus subtilis neutral protease and the neutral protease from Aeromonas proteolytica. Ester hydrolysis by zinc neutral proteases follows the characteristic preference for hydrophobic amino acids adjacent to the site of cleavage, discerned from the hydrolysis of peptide substrates. Removal of zinc from thermolysin abolishes the esterase activity of the native enzyme. Among the metals examined, only Co2+ and Zn2+ restore esterase activity to any significant extent, Co2+ restoring 50% and Zn2+ 100% of the native thermolysin activity. The hydrolysis of esters and peptides by thermolysin does not differ with respect to either the binding or catalytic steps. Substrate specificity, pH-rate profiles, inhibitor, and deuterium isotope effects are identical for both types of substrates. | 2,276 |
pubmed23n0001_1148 | The binding of reduced nicotinamide adenine dinucleotide to citrate synthase of Escherichia coli K12. | Citrate synthase from Escherichia coli enhances the fluorescence of its allosteric inhibitor, NADH, and shifts the peak of emission of the coenzyme from 457 to 428 nm. These effects have been used to measure the binding of NADH to this enzyme under various conditions. The dissociation constant for the NADH-citrate synthase complex is about 0.28 muM at pH 6.2, but increases toward alkaline pH as if binding depends on protonation of a group with a pKa of about 7.05. Over the pH range 6.2-8.7, the number of binding sites decreases from about 0.65 to about 0.25 per citrate synthase subunit. The midpoint of this transition is at about pH 7.7, and it may be one reflection of the partial depolymerization of the enzyme which is known to occur in this pH range. A gel filtration method has been used to verify that the fluorescence enhancement technique accurately reveals all of the NADH molecules bound to the enzyme in the concentration range of interest. NAD+ and NADP+ were weak competitive inhibitors of NADH binding at pH 7.8 (Ki values greater than 1 mM), but stronger inhibition was shown by 5'-AMP and 3'-AMP, with Ki values of 83 +/- 5 and 65 +/- 4 muM, respectively. Acetyl-CoA, one of the substrates, and KCl, an activator, also inhibit the binding in a weakly cooperative manner. All of these effects are consistent with kinetic observations on this system. We interpret our results in terms of two types of binding site for nucleotides on citrate synthase: an active site which binds acetyl-CoA, the substrate, or its analogue 3'-AMP; and an allosteric site which binds NADH or its analogue 5'-AMP and has a lesser affinity for other nicotinamide adenine dinucloetides. When the active site is occupied, we propose that NADH cannot bind to the allosteric site, but 5'-AMP can; conversely, when NADH is the in the allosteric site, the active site cannot be occupied. In addition to these two classes of sites, there must be points for interaction with KCl and other salts. Oxaloacetate, the second substrate, and alpha-ketoglutarate, an inhibitor whose mode of action is believed to be allosteric, have no effect on NADH binding to citrate synthase at pH 7.8. When NADH is bound to citrate synthase, it quenches the intrinsic tryptophan fluorescence of the enzyme. The amount of quenching is proportional to the amount of NADH bound, at least up to a binding ratio of 0.50 NADH per enzyme subunit. This amount of binding leads to the quenching of 53 +/- 5% of the enzyme fluorescence, which means that one NADH molecule can quench all the intrinsic fluorescence of the subunit to which it binds. | The binding of reduced nicotinamide adenine dinucleotide to citrate synthase of Escherichia coli K12. Citrate synthase from Escherichia coli enhances the fluorescence of its allosteric inhibitor, NADH, and shifts the peak of emission of the coenzyme from 457 to 428 nm. These effects have been used to measure the binding of NADH to this enzyme under various conditions. The dissociation constant for the NADH-citrate synthase complex is about 0.28 muM at pH 6.2, but increases toward alkaline pH as if binding depends on protonation of a group with a pKa of about 7.05. Over the pH range 6.2-8.7, the number of binding sites decreases from about 0.65 to about 0.25 per citrate synthase subunit. The midpoint of this transition is at about pH 7.7, and it may be one reflection of the partial depolymerization of the enzyme which is known to occur in this pH range. A gel filtration method has been used to verify that the fluorescence enhancement technique accurately reveals all of the NADH molecules bound to the enzyme in the concentration range of interest. NAD+ and NADP+ were weak competitive inhibitors of NADH binding at pH 7.8 (Ki values greater than 1 mM), but stronger inhibition was shown by 5'-AMP and 3'-AMP, with Ki values of 83 +/- 5 and 65 +/- 4 muM, respectively. Acetyl-CoA, one of the substrates, and KCl, an activator, also inhibit the binding in a weakly cooperative manner. All of these effects are consistent with kinetic observations on this system. We interpret our results in terms of two types of binding site for nucleotides on citrate synthase: an active site which binds acetyl-CoA, the substrate, or its analogue 3'-AMP; and an allosteric site which binds NADH or its analogue 5'-AMP and has a lesser affinity for other nicotinamide adenine dinucloetides. When the active site is occupied, we propose that NADH cannot bind to the allosteric site, but 5'-AMP can; conversely, when NADH is the in the allosteric site, the active site cannot be occupied. In addition to these two classes of sites, there must be points for interaction with KCl and other salts. Oxaloacetate, the second substrate, and alpha-ketoglutarate, an inhibitor whose mode of action is believed to be allosteric, have no effect on NADH binding to citrate synthase at pH 7.8. When NADH is bound to citrate synthase, it quenches the intrinsic tryptophan fluorescence of the enzyme. The amount of quenching is proportional to the amount of NADH bound, at least up to a binding ratio of 0.50 NADH per enzyme subunit. This amount of binding leads to the quenching of 53 +/- 5% of the enzyme fluorescence, which means that one NADH molecule can quench all the intrinsic fluorescence of the subunit to which it binds. | 2,277 |
pubmed23n0001_1149 | The structure of the covalent adduct formed by the interaction of 3-dimethylamino-1-propyne and the flavine of mitochondrial amine oxidase. | 3-Dimethylamino-1-propyne irreversibly inactivates mitochondrial monoamine oxidase from bovine liver. The inactivation results in the loss of absorption in the 450-500-nm region of the flavine spectrum and a concomitant increase in absorbance at 410 nm. For the enzyme-bound adduct epsilon410 = 28000. The spectral properties of the adduct of the liver enzyme with 3-dimethylamino-1-propyne are similar to those observed when the pig kidney enzyme is inactivated with pargyline (Chuang et al. (1974), J. Biol. Chem. 249, 2381). From a proteolytic digest of the enzyme inactivated with labeled inhibitor a flavine peptide has been isolated which contains 1 mol of inactivator/mol of flavine. The chemical and spectral properties of the adduct are those of compounds containing the structure --N--CH==CH--CH==N+ less than. It was concluded that the flavine-inhibtor adduct is a N-5 substituted dihydroflavine and its structure has been determined. | The structure of the covalent adduct formed by the interaction of 3-dimethylamino-1-propyne and the flavine of mitochondrial amine oxidase. 3-Dimethylamino-1-propyne irreversibly inactivates mitochondrial monoamine oxidase from bovine liver. The inactivation results in the loss of absorption in the 450-500-nm region of the flavine spectrum and a concomitant increase in absorbance at 410 nm. For the enzyme-bound adduct epsilon410 = 28000. The spectral properties of the adduct of the liver enzyme with 3-dimethylamino-1-propyne are similar to those observed when the pig kidney enzyme is inactivated with pargyline (Chuang et al. (1974), J. Biol. Chem. 249, 2381). From a proteolytic digest of the enzyme inactivated with labeled inhibitor a flavine peptide has been isolated which contains 1 mol of inactivator/mol of flavine. The chemical and spectral properties of the adduct are those of compounds containing the structure --N--CH==CH--CH==N+ less than. It was concluded that the flavine-inhibtor adduct is a N-5 substituted dihydroflavine and its structure has been determined. | 2,278 |
pubmed23n0001_1150 | The absolute configuration of the amino acids in delta-(alpha-aminoadipyl)cysteinylvaline from Penicillium chrysogenum. | Radioactive carbon-14 L-alpha-aminoadipic acid, L-cysteine, or L-valine were readily incorporated into the intracellular tripeptide, delta-(alpha-aminoadipyl)cysteinylvaline (ACV), by washed starved cells of Penicillium chrysogenum. The labeled ACV in each case was oxidized with performic acid and isolated as its corresponding sulfonic acid derivative. After acid hydrolysis, the configuration of the component acids was determined by L- and D-amino acid oxidases, which showed the tripeptide (ACV) from P. chrysogenum to be delta-(L-aminoadipyl)-L-cysteinyl-D-valine. | The absolute configuration of the amino acids in delta-(alpha-aminoadipyl)cysteinylvaline from Penicillium chrysogenum. Radioactive carbon-14 L-alpha-aminoadipic acid, L-cysteine, or L-valine were readily incorporated into the intracellular tripeptide, delta-(alpha-aminoadipyl)cysteinylvaline (ACV), by washed starved cells of Penicillium chrysogenum. The labeled ACV in each case was oxidized with performic acid and isolated as its corresponding sulfonic acid derivative. After acid hydrolysis, the configuration of the component acids was determined by L- and D-amino acid oxidases, which showed the tripeptide (ACV) from P. chrysogenum to be delta-(L-aminoadipyl)-L-cysteinyl-D-valine. | 2,279 |
pubmed23n0001_1151 | Chromatin-bound protease: degradation of chromosomal proteins under chromatin dissociation conditions. | A chromatin-bound protease, active in 2 M NaCl-5 M urea or 5 M urea alone, was demonstrated in rat liver, kidney, testes, brain, rabbit bone marrow, chicken reticulocyte, and Ehrlich ascites chromatin. Chicken erythrocyte chromatin did not possess any detectable proteolytic activity in salt and urea. The proteolytic activity of rat liver chromatin in salt and urea was found to be independent of the methods of chromatin preparation. The protease can be inhibited by the serine specific reagents phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate and the alkylating reagent, carbobenzoxyphenylalanine chloromethyl ketone, in the presence of organic solvents at 1 mM concentration. The inhibitions of chromatin-bound protease in rat liver by these compounds are irreversible. On the other hand, carbobenzoxyphenylalanine and p-nitrophenyl acetate were shown to be reversible inhibitors of rat liver chromatin-bound protease. The application of these inhibitors during the dissociation of chromatin by salt and urea may be useful to researchers interested in purifying various chromosomal proteins or to those researchers doing reconstitution studies with labile chromatins. | Chromatin-bound protease: degradation of chromosomal proteins under chromatin dissociation conditions. A chromatin-bound protease, active in 2 M NaCl-5 M urea or 5 M urea alone, was demonstrated in rat liver, kidney, testes, brain, rabbit bone marrow, chicken reticulocyte, and Ehrlich ascites chromatin. Chicken erythrocyte chromatin did not possess any detectable proteolytic activity in salt and urea. The proteolytic activity of rat liver chromatin in salt and urea was found to be independent of the methods of chromatin preparation. The protease can be inhibited by the serine specific reagents phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate and the alkylating reagent, carbobenzoxyphenylalanine chloromethyl ketone, in the presence of organic solvents at 1 mM concentration. The inhibitions of chromatin-bound protease in rat liver by these compounds are irreversible. On the other hand, carbobenzoxyphenylalanine and p-nitrophenyl acetate were shown to be reversible inhibitors of rat liver chromatin-bound protease. The application of these inhibitors during the dissociation of chromatin by salt and urea may be useful to researchers interested in purifying various chromosomal proteins or to those researchers doing reconstitution studies with labile chromatins. | 2,280 |
pubmed23n0001_1152 | ATPase of Escherichia coli: purification, dissociation, and reconstitution of the active complex from the isolated subunits. | A simple procedure for the purification of Mg2+-stimulated ATPase of Escherichia coli by fractionation with poly(ethylene glycols) and gel filtration is described. The enzyme restores ATPase-linked reactions to membrane preparations lacking these activities. Five different polypeptides (alpha, beta, gamma, delta, epsilon) are observed in sodium dodecyl sulfate electrophoresis. Freezing in salt solutions splits the enzyme complex into subunits which do not possess any catalytic activity. The presence of different subunits is confirmed by electrophoretic and immunological methods. The active enzyme complex can be reconstituted by decreasing the ionic strength in the dissociated sample. Temperature, pH, protein concentration, and the presence of substrate are each important determinants of the rate and extent of reconstitution. The dissociated enzyme has been separated by ion-exchange chromatography into two major fragments. Fragment IA has a molecular weight of about 100000 and contains the alpha, gamma, and epsilon polypeptides. The minor fragment, IB, has about the same molecular weight but contains, besides alpha, gamma, and epsilon, the delta polypeptide. Fragment II, with a molecular weight of about 52000, appears to be identical with the beta polypeptide. ATPase activity can be reconstituted from fragments IA and II, whereas the capacity of the ATPase to drive energy-dependent processes in depleted membrane vesicles is only restored after incubation of these two fractions with fraction IB, which contains the delta subunit. | ATPase of Escherichia coli: purification, dissociation, and reconstitution of the active complex from the isolated subunits. A simple procedure for the purification of Mg2+-stimulated ATPase of Escherichia coli by fractionation with poly(ethylene glycols) and gel filtration is described. The enzyme restores ATPase-linked reactions to membrane preparations lacking these activities. Five different polypeptides (alpha, beta, gamma, delta, epsilon) are observed in sodium dodecyl sulfate electrophoresis. Freezing in salt solutions splits the enzyme complex into subunits which do not possess any catalytic activity. The presence of different subunits is confirmed by electrophoretic and immunological methods. The active enzyme complex can be reconstituted by decreasing the ionic strength in the dissociated sample. Temperature, pH, protein concentration, and the presence of substrate are each important determinants of the rate and extent of reconstitution. The dissociated enzyme has been separated by ion-exchange chromatography into two major fragments. Fragment IA has a molecular weight of about 100000 and contains the alpha, gamma, and epsilon polypeptides. The minor fragment, IB, has about the same molecular weight but contains, besides alpha, gamma, and epsilon, the delta polypeptide. Fragment II, with a molecular weight of about 52000, appears to be identical with the beta polypeptide. ATPase activity can be reconstituted from fragments IA and II, whereas the capacity of the ATPase to drive energy-dependent processes in depleted membrane vesicles is only restored after incubation of these two fractions with fraction IB, which contains the delta subunit. | 2,281 |
pubmed23n0001_1153 | Conformation of gonadotropin releasing hormone. | The conformation of the gonadotropin releasing hormone (Gn-RH), whose primary sequence is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2, and of several of its structural analogues has been studied by circular dichroism, optical rotatory dispersion, and fluorescence spectroscopy. The effects of pH, guanidine, and temperature on fluorescence emission have also been examined. Titration data demonstrate that the histidine and tyrosine residues are free of any mutual interactions. The similarity of emission spectra in water and in guanidine hydrochloride solutions precludes significant interactions between the fluorescent groups and other residues. Neither the temperature nor the pH profiles of the emission intensities of either tyrosine or tryptophan reveal any fixed secondary structure in Gn-RH. Both the extent of alkaline quenching and the distance of 10-11 A calculated from Förster energy transfer theory are in accord with a randomly coiled structure with only one residue between tyrosine and tryptophan. Furthermore, the circular dichroism spectrum and optical rotatory dispersion do not exhibit any contributions from peptide bonds in an ordered structure, although there is a perturbation of the peptide absorption region due to overlapping bands from side-chain chromophores. Gn-RH, therefore, appears to behave as a random coil polypeptide in water devoid of any intrachain residue interactions. This nonordered structure in Gn-RH and the lack of any significant differences in the physical-chemical properties of the hormone analogues indicate that a predetermined solution conformation is not required for biological activity. In contrast to its behavior in water, Gn-RH in trifluoroethanol exhibits a conformational transition, with the formation of a beta structure. Differences in conformational changes exhibited by several analogues in trifluoroethanol may be relevant to their relative biological activities at the receptor site. | Conformation of gonadotropin releasing hormone. The conformation of the gonadotropin releasing hormone (Gn-RH), whose primary sequence is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2, and of several of its structural analogues has been studied by circular dichroism, optical rotatory dispersion, and fluorescence spectroscopy. The effects of pH, guanidine, and temperature on fluorescence emission have also been examined. Titration data demonstrate that the histidine and tyrosine residues are free of any mutual interactions. The similarity of emission spectra in water and in guanidine hydrochloride solutions precludes significant interactions between the fluorescent groups and other residues. Neither the temperature nor the pH profiles of the emission intensities of either tyrosine or tryptophan reveal any fixed secondary structure in Gn-RH. Both the extent of alkaline quenching and the distance of 10-11 A calculated from Förster energy transfer theory are in accord with a randomly coiled structure with only one residue between tyrosine and tryptophan. Furthermore, the circular dichroism spectrum and optical rotatory dispersion do not exhibit any contributions from peptide bonds in an ordered structure, although there is a perturbation of the peptide absorption region due to overlapping bands from side-chain chromophores. Gn-RH, therefore, appears to behave as a random coil polypeptide in water devoid of any intrachain residue interactions. This nonordered structure in Gn-RH and the lack of any significant differences in the physical-chemical properties of the hormone analogues indicate that a predetermined solution conformation is not required for biological activity. In contrast to its behavior in water, Gn-RH in trifluoroethanol exhibits a conformational transition, with the formation of a beta structure. Differences in conformational changes exhibited by several analogues in trifluoroethanol may be relevant to their relative biological activities at the receptor site. | 2,282 |
pubmed23n0001_1154 | Low and high pH form of cadmium carbonic anhydrase determined by nuclear quadrupole interaction. | The pH dependence of the nuclear quadrupole interaction between the excited 247-keV state in 111Cd bound to the active site in human carbonic anhydrase B and the nearest protein surroundings has been studied by means of the nuclear spectroscopic technique of perturbed angular correlation of gamma rays. The enzyme has been studied in the pH region 5.6-11.0 at 22 and -196 degrees C. The results show that the Cd enzyme changes from one form at low pH to another form at high pH both at 22 and -196 degrees C. The pK of the transition is 8.9 +/- 0.2 at -196 degrees C and close to 9 at 22 degrees C. Parallel to this transformation, the esterase activity of the Cd enzyme for the hydration of p-nitrophenyl acetate exhibits a pH dependency with a pH of 9.1 +/- 0.2. The sulfonamide inhibitor acetazolamide completely inhibits this activity of the Cd enzyme. The quadrupole interaction parameters for the Cd enzyme are not significantly different at -196 degrees C from those obtained at 22 degrees C. A measurement at 0 degrees C pH 5.7 shows, however, a form different from those at 22 degrees C pH 5.6 and -196 degrees C pH 5.7. The change in the quadrupole interaction with pH is, in a simple model, consistent with an ionization of a metal-bound water molecule. | Low and high pH form of cadmium carbonic anhydrase determined by nuclear quadrupole interaction. The pH dependence of the nuclear quadrupole interaction between the excited 247-keV state in 111Cd bound to the active site in human carbonic anhydrase B and the nearest protein surroundings has been studied by means of the nuclear spectroscopic technique of perturbed angular correlation of gamma rays. The enzyme has been studied in the pH region 5.6-11.0 at 22 and -196 degrees C. The results show that the Cd enzyme changes from one form at low pH to another form at high pH both at 22 and -196 degrees C. The pK of the transition is 8.9 +/- 0.2 at -196 degrees C and close to 9 at 22 degrees C. Parallel to this transformation, the esterase activity of the Cd enzyme for the hydration of p-nitrophenyl acetate exhibits a pH dependency with a pH of 9.1 +/- 0.2. The sulfonamide inhibitor acetazolamide completely inhibits this activity of the Cd enzyme. The quadrupole interaction parameters for the Cd enzyme are not significantly different at -196 degrees C from those obtained at 22 degrees C. A measurement at 0 degrees C pH 5.7 shows, however, a form different from those at 22 degrees C pH 5.6 and -196 degrees C pH 5.7. The change in the quadrupole interaction with pH is, in a simple model, consistent with an ionization of a metal-bound water molecule. | 2,284 |
pubmed23n0001_1155 | Oxidation-reduction properties of Chromatium vinosum high potential iron-sulfur protein. | The oxidation-reduction properties of the high potential iron-sulfur protein (HIPIP) from Chromatium vinosum have been investigated. Both equilibrium and kinetic measurements demonstrate electron transport by HIPIP is pH independent in the pH range 7-11. The kinetics of reduction (potassium ferrocyanide, SO2, S2O42-, sodium ascorbate, and Rhodospirillum rubrum cytochrome c2) and oxidation (potassium ferricyanide and Rhodospirillium rubrum cytochrome c2) of HIPIP are reported. Based on the data obtained with different reactants and the influence of ionic strength, pH, and temperature on the kinetics of oxidation and reduction, a number of conclusions can be drawn. (1) HIPIP undergoes rapid outer-sphere electron transfer with no evidence of kinetic complexity and no indication of complex formation with various reactants. (2) The site of oxidation of reduced HIPIP has an apparent negative charge while the site of reduction of oxidized HIPIP is uncharged. (3) HIPIP appears to interact with a physiological reactant (R. rubrum cytochrome c2) at the same site as nonphysiological oxidants or reductants suggesting single minimum energy pathways for the oxidation and reduction processes. (4) Based on a comparison of the rates of oxidation and reduction with different reactants, it appears that steric restrictions and differences in oxidation-reduction potential are less important than electrostatic attraction and/or repulsion in determining the absolute rate constants. (5) The thermodynamic activation parameters indicate that both oxidation and reduction by the iron hexacyanides are driven entropically with the enthalpic terms making no contribution to HIPIP oxidation and a small contribution to HIPIP reduction. Based on the data reported here and available structural and physical-chemical information, possible mechanisms of the oxidation and reduction of HIPIP are discussed and their relative merits analyzed. The more likely mechanisms include electron transfer via a tyrosine residue, electron transfer through a nonaqueous media to the iron-sulfur chromophore, and direct interaction between the iron-sulfur chromophore and the different oxidants and reductants. | Oxidation-reduction properties of Chromatium vinosum high potential iron-sulfur protein. The oxidation-reduction properties of the high potential iron-sulfur protein (HIPIP) from Chromatium vinosum have been investigated. Both equilibrium and kinetic measurements demonstrate electron transport by HIPIP is pH independent in the pH range 7-11. The kinetics of reduction (potassium ferrocyanide, SO2, S2O42-, sodium ascorbate, and Rhodospirillum rubrum cytochrome c2) and oxidation (potassium ferricyanide and Rhodospirillium rubrum cytochrome c2) of HIPIP are reported. Based on the data obtained with different reactants and the influence of ionic strength, pH, and temperature on the kinetics of oxidation and reduction, a number of conclusions can be drawn. (1) HIPIP undergoes rapid outer-sphere electron transfer with no evidence of kinetic complexity and no indication of complex formation with various reactants. (2) The site of oxidation of reduced HIPIP has an apparent negative charge while the site of reduction of oxidized HIPIP is uncharged. (3) HIPIP appears to interact with a physiological reactant (R. rubrum cytochrome c2) at the same site as nonphysiological oxidants or reductants suggesting single minimum energy pathways for the oxidation and reduction processes. (4) Based on a comparison of the rates of oxidation and reduction with different reactants, it appears that steric restrictions and differences in oxidation-reduction potential are less important than electrostatic attraction and/or repulsion in determining the absolute rate constants. (5) The thermodynamic activation parameters indicate that both oxidation and reduction by the iron hexacyanides are driven entropically with the enthalpic terms making no contribution to HIPIP oxidation and a small contribution to HIPIP reduction. Based on the data reported here and available structural and physical-chemical information, possible mechanisms of the oxidation and reduction of HIPIP are discussed and their relative merits analyzed. The more likely mechanisms include electron transfer via a tyrosine residue, electron transfer through a nonaqueous media to the iron-sulfur chromophore, and direct interaction between the iron-sulfur chromophore and the different oxidants and reductants. | 2,285 |
pubmed23n0001_1156 | Isolation, chemical, and physical properties of alpha-1-antitrypsin. | A method of isolation of alpha-1-antitrypsin (alpha-1-AT) in good yield from normal human plasma is described. A key step was affinity chromatography employing an antiserum which had been depleted of alpha-1-AT antibodies. The final preparations were homogeneous by immunological and physicochemical criteria. The specific activity of the purified alpha-1-AT was 0.363 mg of active bovine trypsin inhibited per 1.0 mg of inhibitor. Polyacrylamide gel patterns at both alkaline and acid pH of highly pure preparations frequently, but not invariably, showed multiple hands. Molecular weight studies by sedimentation equilibrium ultracentrifugation in aqueous buffer and in 6 M guanidine as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis suggest that alpha-1-AT is a single polypeptide chain having a molecular weight of 49,500. Other physical and chemical properties of the inhibitor are described. A limited N-terminal sequence (Glu-Asp-Pro-Gln-Gly-Asx-Ala-Ala) was obtained. It was found that alpha-1-AT easily forms polymers and higher aggregates when exposed to denaturing agents such as 8 M urea and 6 M guanidine. The results suggest that aggregation is determined by both covalent and noncovalent forces. | Isolation, chemical, and physical properties of alpha-1-antitrypsin. A method of isolation of alpha-1-antitrypsin (alpha-1-AT) in good yield from normal human plasma is described. A key step was affinity chromatography employing an antiserum which had been depleted of alpha-1-AT antibodies. The final preparations were homogeneous by immunological and physicochemical criteria. The specific activity of the purified alpha-1-AT was 0.363 mg of active bovine trypsin inhibited per 1.0 mg of inhibitor. Polyacrylamide gel patterns at both alkaline and acid pH of highly pure preparations frequently, but not invariably, showed multiple hands. Molecular weight studies by sedimentation equilibrium ultracentrifugation in aqueous buffer and in 6 M guanidine as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis suggest that alpha-1-AT is a single polypeptide chain having a molecular weight of 49,500. Other physical and chemical properties of the inhibitor are described. A limited N-terminal sequence (Glu-Asp-Pro-Gln-Gly-Asx-Ala-Ala) was obtained. It was found that alpha-1-AT easily forms polymers and higher aggregates when exposed to denaturing agents such as 8 M urea and 6 M guanidine. The results suggest that aggregation is determined by both covalent and noncovalent forces. | 2,286 |
pubmed23n0001_1157 | Purification and properties of the thermostable acid protease of Penicillium duponti. | An acid protease produced by the thermophilic fungus Penicillium duponti K 1014 has been purified by consecutive ion-exchange and gel permeation chromatography, and crystallized from aqueous acetone solution. The purified endopeptidase gave a symmetrical schlieren peak by sedimentation velocity, and was found to be homogeneous upon disc gel electrophoresis at pH 9.5. The enzyme was most active at pH 2.5 against milk casein and showed high thermostability. An isoelectric point of 3.81 was found by isoelectric focusing. A minimum molecular weight of 41 590 was calculated from the amino acid composition, adopting an arginine content of one residue per mole of enzyme. This minimum molecular weight is in good agreement with the value of 41 000 previously found by gel permeation (Hashimoto, H., Iwaasa, T., and Yokotsuka, T. (1973), Appl. Microbiol. 25, 578). Besides the thermostability, the purified P. duponti protease differs from other well-characterized acid proteases in that it contains carbohydrate, 4.33% expressed as glucose. The enzyme was not affected by p-bromophenacyl bromide, but was completely inactivated by alpha-diazo-p-bromoacetophenone, diazoacetyl-DL-norleucine methyl ester, and diazoacetylglycine ethyl ester, in the presence of Cu2+. The complete inactivation of the protease by diazoacetyl-DL-norleucine methyl ester resulted in the specific incorporation of 1 mol of norleucine/mol of enzyme. On the basis of similar behavior of other acid proteases toward this inactivator, the results suggest the presence at the active site of an unusually reactive carboxyl group, involved in the catalytic function. The naturally occurring pepsin inhibitor of Streptomyces naniwaensis [Murao, S., and Satoi, S. (1970), Agric. Biol. Chem. 34, 1265] inhibited also the protease, at a threefold molar excess with respect to the enzyme. | Purification and properties of the thermostable acid protease of Penicillium duponti. An acid protease produced by the thermophilic fungus Penicillium duponti K 1014 has been purified by consecutive ion-exchange and gel permeation chromatography, and crystallized from aqueous acetone solution. The purified endopeptidase gave a symmetrical schlieren peak by sedimentation velocity, and was found to be homogeneous upon disc gel electrophoresis at pH 9.5. The enzyme was most active at pH 2.5 against milk casein and showed high thermostability. An isoelectric point of 3.81 was found by isoelectric focusing. A minimum molecular weight of 41 590 was calculated from the amino acid composition, adopting an arginine content of one residue per mole of enzyme. This minimum molecular weight is in good agreement with the value of 41 000 previously found by gel permeation (Hashimoto, H., Iwaasa, T., and Yokotsuka, T. (1973), Appl. Microbiol. 25, 578). Besides the thermostability, the purified P. duponti protease differs from other well-characterized acid proteases in that it contains carbohydrate, 4.33% expressed as glucose. The enzyme was not affected by p-bromophenacyl bromide, but was completely inactivated by alpha-diazo-p-bromoacetophenone, diazoacetyl-DL-norleucine methyl ester, and diazoacetylglycine ethyl ester, in the presence of Cu2+. The complete inactivation of the protease by diazoacetyl-DL-norleucine methyl ester resulted in the specific incorporation of 1 mol of norleucine/mol of enzyme. On the basis of similar behavior of other acid proteases toward this inactivator, the results suggest the presence at the active site of an unusually reactive carboxyl group, involved in the catalytic function. The naturally occurring pepsin inhibitor of Streptomyces naniwaensis [Murao, S., and Satoi, S. (1970), Agric. Biol. Chem. 34, 1265] inhibited also the protease, at a threefold molar excess with respect to the enzyme. | 2,287 |
pubmed23n0001_1158 | Biosynthesis of bacterial glycogen. Purification and properties of the Escherichia coli B ADPglucose:1,4-alpha-D-glucan 4-alpha-glucosyltransferase. | The Escherichia coli B glycogen synthase has been purified to apparent homogeneity with the use of a 4-aminobutyl-Sepharose column. Two fractions of the enzyme were obtained: glycogen synthase I with a specific activity of 380 mumol mg-1 and devoid of branching enzyme activity and glycogen synthase II having a specific activity of 505 mumol mg-1 and containing branching enzyme activity which was 0.1% of the activity observed for the glycogen synthase. Only one protein band was found in disc gel electrophoresis for each glycogen synthase fraction and they were coincident with glycogen synthase activity. One major protein band and one very faint protein band which hardly moved into the gel were observed in sodium dodecyl sulfate gel electrophoresis of the glycogen synthase fractions. The subunit molecular weight of the major protein band in sodium dodecyl sulfate gel electrophoresis of both glycogen synthase fractions was determined to be 49 000 +/- 2 000. The molecular weights of the native enzymes were determined by sucrose density gradient ultracentrifugation. Glycogen synthase I had a molecular weight of 93 000 while glycogen synthase II had a molecular weight of 200 000. On standing at 4 degrees C or at -85 degrees C both enzymes transform into species having molecular weights of 98 000, 135 000, and 185 000. Thus active forms of the E. coli B glycogen synthase can exist as dimers, trimers, and tetramers of the subunit. The enzyme was shown to catalyze transfer of glucose from ADPglucose to maltose and to higher oligosaccharides of the maltodextrin series but not to glucose. 1,5-Gluconolactone was shown to be a potent inhibitor of the glycogen synthase reaction. The glycogen synthase reaction was shown to be reversible. Formation of labeled ADPglucose occurred from either [14C]ADP or [14C]glycogen. The ratio of ADP to ADPglucose at equilibrium at 37 degrees C was determined and was found to vary threefold in the pH range of 5.27-6.82. From these data the ratio of ADP2- to ADPglucose at equilibrium was determined to be 45.8 +/- 4.5. Assuming that deltaF degrees of the hydrolysis of the alpha-1,4-glucosidic linkage is -4.0 kcal the deltaF degrees of hydrolysis of the glucosidic linkage in ADPglucose is -6.3 kcal. | Biosynthesis of bacterial glycogen. Purification and properties of the Escherichia coli B ADPglucose:1,4-alpha-D-glucan 4-alpha-glucosyltransferase. The Escherichia coli B glycogen synthase has been purified to apparent homogeneity with the use of a 4-aminobutyl-Sepharose column. Two fractions of the enzyme were obtained: glycogen synthase I with a specific activity of 380 mumol mg-1 and devoid of branching enzyme activity and glycogen synthase II having a specific activity of 505 mumol mg-1 and containing branching enzyme activity which was 0.1% of the activity observed for the glycogen synthase. Only one protein band was found in disc gel electrophoresis for each glycogen synthase fraction and they were coincident with glycogen synthase activity. One major protein band and one very faint protein band which hardly moved into the gel were observed in sodium dodecyl sulfate gel electrophoresis of the glycogen synthase fractions. The subunit molecular weight of the major protein band in sodium dodecyl sulfate gel electrophoresis of both glycogen synthase fractions was determined to be 49 000 +/- 2 000. The molecular weights of the native enzymes were determined by sucrose density gradient ultracentrifugation. Glycogen synthase I had a molecular weight of 93 000 while glycogen synthase II had a molecular weight of 200 000. On standing at 4 degrees C or at -85 degrees C both enzymes transform into species having molecular weights of 98 000, 135 000, and 185 000. Thus active forms of the E. coli B glycogen synthase can exist as dimers, trimers, and tetramers of the subunit. The enzyme was shown to catalyze transfer of glucose from ADPglucose to maltose and to higher oligosaccharides of the maltodextrin series but not to glucose. 1,5-Gluconolactone was shown to be a potent inhibitor of the glycogen synthase reaction. The glycogen synthase reaction was shown to be reversible. Formation of labeled ADPglucose occurred from either [14C]ADP or [14C]glycogen. The ratio of ADP to ADPglucose at equilibrium at 37 degrees C was determined and was found to vary threefold in the pH range of 5.27-6.82. From these data the ratio of ADP2- to ADPglucose at equilibrium was determined to be 45.8 +/- 4.5. Assuming that deltaF degrees of the hydrolysis of the alpha-1,4-glucosidic linkage is -4.0 kcal the deltaF degrees of hydrolysis of the glucosidic linkage in ADPglucose is -6.3 kcal. | 2,288 |
pubmed23n0001_1159 | Bovine procarboxypeptidase A: kinetics of peptide and ester hydrolysis. | Bovine procarboxypeptidase A exhibits intrinsic hydrolytic activity toward haloacyl amino acids (Behnke and Vallee, 1972), as well as toward conventional peptide and ester substrates for carboxypeptidase A (Bezzone, 1974; Uren and Neurath, 1974). The kinetics of hydrolysis of a series of such substrates by native procarboxypeptidase has now been examined in detail in order to ascertain the extent to which the binding and catalytic sites of carboxypeptidase preexist inthe zymogen. Distinct differences in the substrate binding sites of the zymogen compared with the enzyme are apparent from their respective kinetic profiles as well as from the effects of modifiers on their activities. Substrate activation with the dipeptides BzGly-L-Phe and CbzGly-L-Phe, well known for carboxypeptidase, is exhibited also by the zymogen, but the corresponding substrate inhibition by CbzGly-L-Phe and BzGly-Ophe is absent. Moreover, the substrate inhibition of carboxypeptidase by CbzGlyGly-L-Phe and BzGly-Ophe is replaced by substrate activation in the zymogen... | Bovine procarboxypeptidase A: kinetics of peptide and ester hydrolysis. Bovine procarboxypeptidase A exhibits intrinsic hydrolytic activity toward haloacyl amino acids (Behnke and Vallee, 1972), as well as toward conventional peptide and ester substrates for carboxypeptidase A (Bezzone, 1974; Uren and Neurath, 1974). The kinetics of hydrolysis of a series of such substrates by native procarboxypeptidase has now been examined in detail in order to ascertain the extent to which the binding and catalytic sites of carboxypeptidase preexist inthe zymogen. Distinct differences in the substrate binding sites of the zymogen compared with the enzyme are apparent from their respective kinetic profiles as well as from the effects of modifiers on their activities. Substrate activation with the dipeptides BzGly-L-Phe and CbzGly-L-Phe, well known for carboxypeptidase, is exhibited also by the zymogen, but the corresponding substrate inhibition by CbzGly-L-Phe and BzGly-Ophe is absent. Moreover, the substrate inhibition of carboxypeptidase by CbzGlyGly-L-Phe and BzGly-Ophe is replaced by substrate activation in the zymogen... | 2,289 |
pubmed23n0001_1160 | Ionic influences on the phase transition of dipalmitoylphosphatidylserine. | The ionization and phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine have been investigated under a variety of condtions by several different methods. As measured by turbidity changes, the temperature of the crystal-liquid crystal phase transition of this lipid is influenced by pH and mono- and divalent cation concentrations. The pH-transition temperature curve is congruent with the curve relating temperature to the degree of ionization of the carboxyl group of the crystalline form. The transition temperature falls from an upper plateau of 72 degrees C at low pH values, where the carboxyl group is fully protonated, to a lower plateau of 55 degrees C at high pH values, where this group is fully ionized. The apparent pK (pH at 50% ionization) of the crystalline form shifts from 6.0 to 4.6 to 3.7 with an increase of NaCl concentration from 10(-3) to 0.1 to l.0 M, respectively. These observations are in accord with a simple theoretical analysis that utilizes diffuse double layer theory and the influence of surface potential on surface concentration of protons. In qualitative terms, an increase in electrolyte concentration reduces the surface potential, the result of which is a diminution of the surface-bulk pH difference and a lowering of the apparent pK. Assuming an area of 50 A2/molecule, the intrinsic pKa (apparent pK corrected for surface pH) of the carboxyl group is 2.7. A 1000-fold change of NaCl concentration produces a very large change in surface potential without influencing the transition temperature of the ionized form of the lipid. | Ionic influences on the phase transition of dipalmitoylphosphatidylserine. The ionization and phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine have been investigated under a variety of condtions by several different methods. As measured by turbidity changes, the temperature of the crystal-liquid crystal phase transition of this lipid is influenced by pH and mono- and divalent cation concentrations. The pH-transition temperature curve is congruent with the curve relating temperature to the degree of ionization of the carboxyl group of the crystalline form. The transition temperature falls from an upper plateau of 72 degrees C at low pH values, where the carboxyl group is fully protonated, to a lower plateau of 55 degrees C at high pH values, where this group is fully ionized. The apparent pK (pH at 50% ionization) of the crystalline form shifts from 6.0 to 4.6 to 3.7 with an increase of NaCl concentration from 10(-3) to 0.1 to l.0 M, respectively. These observations are in accord with a simple theoretical analysis that utilizes diffuse double layer theory and the influence of surface potential on surface concentration of protons. In qualitative terms, an increase in electrolyte concentration reduces the surface potential, the result of which is a diminution of the surface-bulk pH difference and a lowering of the apparent pK. Assuming an area of 50 A2/molecule, the intrinsic pKa (apparent pK corrected for surface pH) of the carboxyl group is 2.7. A 1000-fold change of NaCl concentration produces a very large change in surface potential without influencing the transition temperature of the ionized form of the lipid. | 2,290 |
pubmed23n0001_1161 | Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria. | The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm. The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory. An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter. The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index. Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP. The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP. | Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria. The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm. The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory. An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter. The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index. Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP. The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP. | 2,291 |
pubmed23n0001_1162 | Isolation and characterization of two alkaline ribonucleases from calf serum. | Treatment of calf serum at 60 degrees C and pH 3.5 followed by chromatography on carboxymethyl (CM) cellulose resulted in the separation of two major peaks of alkaline RNAse activity. One was eluted from CM-cellulose at 0.075 M KCl with an overall purification of 5400-fold and the other was eluted at 0.25 M KCl with a 6700-fold purification. The RNAse eluted from CM-cellulose at 0.075 M KCl was almost completely inhibited by anti-RNAse A serum and by the endogenous RNAse inhibitor and a 33% inhibition was observed in the presence of 5 mM MgCl2. This enzyme seems to be similar or identical to RNAse A. The other RNAse, eluted from CM-cellulose at 0.25 M KCl was not inhibited by anti-RNAse A or 5 mM MgCl2 and was much less sensitive to the endogenous inhibitor. Both enzymes degraded RNA endonucleolytically and the nucleoside monophosphates obtained after partial hydrolysis of RNA by the two serum RNAases were primarily 2'- or 3' -CMP and 2'- or 3' -UMP. Poly(A), native DNA and denatured DNA were degraded slowly or not at all. The RNAase A-like enzyme degraded poly(C) at a significantly faster rate, and poly(U) at a slower rate, than RNA. However, the other serum RNAase was more active with poly(U) than with RNA and almost inactive with poly(C) as the substrate. | Isolation and characterization of two alkaline ribonucleases from calf serum. Treatment of calf serum at 60 degrees C and pH 3.5 followed by chromatography on carboxymethyl (CM) cellulose resulted in the separation of two major peaks of alkaline RNAse activity. One was eluted from CM-cellulose at 0.075 M KCl with an overall purification of 5400-fold and the other was eluted at 0.25 M KCl with a 6700-fold purification. The RNAse eluted from CM-cellulose at 0.075 M KCl was almost completely inhibited by anti-RNAse A serum and by the endogenous RNAse inhibitor and a 33% inhibition was observed in the presence of 5 mM MgCl2. This enzyme seems to be similar or identical to RNAse A. The other RNAse, eluted from CM-cellulose at 0.25 M KCl was not inhibited by anti-RNAse A or 5 mM MgCl2 and was much less sensitive to the endogenous inhibitor. Both enzymes degraded RNA endonucleolytically and the nucleoside monophosphates obtained after partial hydrolysis of RNA by the two serum RNAases were primarily 2'- or 3' -CMP and 2'- or 3' -UMP. Poly(A), native DNA and denatured DNA were degraded slowly or not at all. The RNAase A-like enzyme degraded poly(C) at a significantly faster rate, and poly(U) at a slower rate, than RNA. However, the other serum RNAase was more active with poly(U) than with RNA and almost inactive with poly(C) as the substrate. | 2,292 |
pubmed23n0001_1163 | Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells. | A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveals no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure. | Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells. A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveals no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure. | 2,293 |
pubmed23n0001_1164 | Glial fibrillary acidic protein from bovine and rat brain. Degradation in tissues and homogenates. | Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea. | Glial fibrillary acidic protein from bovine and rat brain. Degradation in tissues and homogenates. Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea. | 2,294 |
pubmed23n0001_1165 | Studies on the binding of haemoglobin by haptoglobin using electrofocusing and gradient electrophoresis. | 1. Gel electrofocusing followed by gel gradient electrophoresis separated the haptoglobins and their complexes with haemoglobin into characteristic two-dimensional patterns of protein bands. 2. Molecular weights of 107 000, 139 000 and 168 000 were obtained for the three bands seen after a purified preparation of haptoglobin type 1 was partially saturated with haemoglobin. This indicated that free haptoglobin, the intermediate haptoglobin-haemoglobin complex containing one half-haemoglobin and the saturated complex with two half-haemoglobins were present. 3. The three proteins showed considerable microheterogeneity and gave a number of isoelectric points in the pH ranges 4.58-4.77, 5.20-5.40 and 5.74-5.93, free haptoglobin type 1 being the lowest group. These ranges were all 0.15-0.30pH units lower if other values were taken for the isoelectric points of markers used to calibrate the pH gradient. 4. All three proteins were present over a wide range of haemoglobin concentrations, from 0.5% to 92% of that required for saturation. This would be expected if both binding sites have similar affinities for haemoglobin. | Studies on the binding of haemoglobin by haptoglobin using electrofocusing and gradient electrophoresis. 1. Gel electrofocusing followed by gel gradient electrophoresis separated the haptoglobins and their complexes with haemoglobin into characteristic two-dimensional patterns of protein bands. 2. Molecular weights of 107 000, 139 000 and 168 000 were obtained for the three bands seen after a purified preparation of haptoglobin type 1 was partially saturated with haemoglobin. This indicated that free haptoglobin, the intermediate haptoglobin-haemoglobin complex containing one half-haemoglobin and the saturated complex with two half-haemoglobins were present. 3. The three proteins showed considerable microheterogeneity and gave a number of isoelectric points in the pH ranges 4.58-4.77, 5.20-5.40 and 5.74-5.93, free haptoglobin type 1 being the lowest group. These ranges were all 0.15-0.30pH units lower if other values were taken for the isoelectric points of markers used to calibrate the pH gradient. 4. All three proteins were present over a wide range of haemoglobin concentrations, from 0.5% to 92% of that required for saturation. This would be expected if both binding sites have similar affinities for haemoglobin. | 2,295 |
pubmed23n0001_1166 | Cytochrome P450cam and its complexes. Mössbauer parameters of the heme iron. | Mössbauer spectroscopy has been used to study the heme iron in various states of cytochrome P450cam from the camphor-hydroxylating system of the bacterium Pseudomonas putida. Native, camphor-free P450cam contains low-spin ferric iron, part of which (approx. 50-70%) is converted to the high-spin ferric state upon addition of camphor. The Mössbauer spectra of the camphor-free enzyme (S equals 1/2) and of the high-spin component (S equals 5/2) of the camphor complex have been successfully simulated using a model based on crystal-field theory and simple convalency considerations. The native low-spin ferric state of P450cam forms a complex with 2-phenylimidazole, with small changes in the g values and Mössbauer spectra. These changes can be accounted for consistently in the crystal-field model referred to above. The addition of putidaredoxin to the camphor-complexed, oxidized P450cam decreases the intensity of the high-spin component and changes its quadrupole splitting. The reduced form of P450cam contrins high-spin ferrous iron, both in the presence and absence of camphor. The complex of reduced P450cam with molecular oxygen is diamagnetic and has a combination of quadrupole splitting and isomer shift that is unusual for a ferrous complex, but strongly resembles that of oxyhemoglobin. These results are compatible with the bound superoxide, Fe3+-O-2, model proposed for oxyhemoglobin (Weiss, J. J. (1964) Nature 202, 83-84). Reduced P450cam and its complexes, oxyP450cam-CO, are all found to be analogous in some respects to the corresponding hemoglobin complexes. | Cytochrome P450cam and its complexes. Mössbauer parameters of the heme iron. Mössbauer spectroscopy has been used to study the heme iron in various states of cytochrome P450cam from the camphor-hydroxylating system of the bacterium Pseudomonas putida. Native, camphor-free P450cam contains low-spin ferric iron, part of which (approx. 50-70%) is converted to the high-spin ferric state upon addition of camphor. The Mössbauer spectra of the camphor-free enzyme (S equals 1/2) and of the high-spin component (S equals 5/2) of the camphor complex have been successfully simulated using a model based on crystal-field theory and simple convalency considerations. The native low-spin ferric state of P450cam forms a complex with 2-phenylimidazole, with small changes in the g values and Mössbauer spectra. These changes can be accounted for consistently in the crystal-field model referred to above. The addition of putidaredoxin to the camphor-complexed, oxidized P450cam decreases the intensity of the high-spin component and changes its quadrupole splitting. The reduced form of P450cam contrins high-spin ferrous iron, both in the presence and absence of camphor. The complex of reduced P450cam with molecular oxygen is diamagnetic and has a combination of quadrupole splitting and isomer shift that is unusual for a ferrous complex, but strongly resembles that of oxyhemoglobin. These results are compatible with the bound superoxide, Fe3+-O-2, model proposed for oxyhemoglobin (Weiss, J. J. (1964) Nature 202, 83-84). Reduced P450cam and its complexes, oxyP450cam-CO, are all found to be analogous in some respects to the corresponding hemoglobin complexes. | 2,296 |
pubmed23n0001_1167 | Cross partition and determination of net charge of the isoenzymes of enolase. | Enolase from bakers' yeast was separated into three isoenzymes by countercurrent distribution. The isoenzymes were partitioned in aqueous polymer two-phase systems containing positively charged trimethylamino poly(ethylene glycol) or negatively charged poly(ethylene glycol) sulphonate. The plots of the partition coefficient of each isoenzyme versus pH in the two biphasic systems intersect at pH equal to the isoelectric point. From slopes of the plots, the net charge of the isoenzymes at pH 6.57 was determined to be +2, -3, and -8 respectively. | Cross partition and determination of net charge of the isoenzymes of enolase. Enolase from bakers' yeast was separated into three isoenzymes by countercurrent distribution. The isoenzymes were partitioned in aqueous polymer two-phase systems containing positively charged trimethylamino poly(ethylene glycol) or negatively charged poly(ethylene glycol) sulphonate. The plots of the partition coefficient of each isoenzyme versus pH in the two biphasic systems intersect at pH equal to the isoelectric point. From slopes of the plots, the net charge of the isoenzymes at pH 6.57 was determined to be +2, -3, and -8 respectively. | 2,297 |
pubmed23n0001_1168 | Avoidance of strongly chaotropic eluents for immunoaffinity chromatography by chemical modification of immobilized ligand. | The need for chaotropic eluents in immunoaffinity chromatography is a consequence of the high affinities of antibodies towards their antigens. This affinity is decreased and elution of antiglucagon antibodies from a column of immobilized glucagon can be achieved under mild conditions when the steric complementarity to the antibody binding site is perturbed by selective chemical modification of the hormone. The effects of reaction with 2-hydroxy-5-nitrobenzyl bromide, tetranitromethane and hydrogen peroxide have been studied. Conversely, treatment of immobilized antibodies with 2-hydroxy-5-nitrobenzyl bromide facilitates the elution of glucagon during immunoaffinity chromatography. The general implications of these results are discussed. | Avoidance of strongly chaotropic eluents for immunoaffinity chromatography by chemical modification of immobilized ligand. The need for chaotropic eluents in immunoaffinity chromatography is a consequence of the high affinities of antibodies towards their antigens. This affinity is decreased and elution of antiglucagon antibodies from a column of immobilized glucagon can be achieved under mild conditions when the steric complementarity to the antibody binding site is perturbed by selective chemical modification of the hormone. The effects of reaction with 2-hydroxy-5-nitrobenzyl bromide, tetranitromethane and hydrogen peroxide have been studied. Conversely, treatment of immobilized antibodies with 2-hydroxy-5-nitrobenzyl bromide facilitates the elution of glucagon during immunoaffinity chromatography. The general implications of these results are discussed. | 2,298 |
pubmed23n0001_1169 | Release and activation of a particulate bound acid phosphatase from Tetrahymena pyriformis. | A sedimentable form of acid phosphatase (EC 3.1.3.2) from Tetrahymena pyriformis was found to be solubilized by Triton X-100. The total enzyme activity in the insoluble cell fraction increased almost 200% upon solubilization with Triton X-100 or Nonidet P-40. Removal of membrane lipids and Triton X-100 from the particulate wash solution with a chloroform extraction resulted in non-specific enzyme-protein aggregation which was reversible upon addition of Triton X-100. The results indicate that this acid phosphatase is an integral membrane protein. The pH optima for this particulate bound acid phosphatase was 3.5 with o-carboxyphenyl phosphate and 4.0 with p-nitrophenyl phosphate as substrates. The Km values of each substrate were 3.1 and 0.031 mM, respectively. | Release and activation of a particulate bound acid phosphatase from Tetrahymena pyriformis. A sedimentable form of acid phosphatase (EC 3.1.3.2) from Tetrahymena pyriformis was found to be solubilized by Triton X-100. The total enzyme activity in the insoluble cell fraction increased almost 200% upon solubilization with Triton X-100 or Nonidet P-40. Removal of membrane lipids and Triton X-100 from the particulate wash solution with a chloroform extraction resulted in non-specific enzyme-protein aggregation which was reversible upon addition of Triton X-100. The results indicate that this acid phosphatase is an integral membrane protein. The pH optima for this particulate bound acid phosphatase was 3.5 with o-carboxyphenyl phosphate and 4.0 with p-nitrophenyl phosphate as substrates. The Km values of each substrate were 3.1 and 0.031 mM, respectively. | 2,299 |
pubmed23n0001_1170 | Partial purification and characterization of post-proline cleaving enzyme: enzymatic inactivation of neurohypophyseal hormones by kidney preparations of various species. | The inactivation of the neurohypophyseal hormones arginine vasopressin and oxytocin, both 14C-labelled in the C-terminal glycine residue, by enzymes present in kidney homogenates of various species has been investigated, and some of the enzymes responsible have been partially purified and characterized. The Leu-Gly peptide bond of oxytocin is generally most effectively cleaved by kidney homogenates, although with certain species enzymic activity hydrolyzing the Pro-Leu bond is significant. Degradation of arginine vasopressin is slower than oxytocin in all species studied, and appears to occur by a different overall mechanism since cleavage of the Pro-Arg bond is more significant than hydrolysis of the Arg-Gly bond. The enzyme releasing glycinamide from oxytocin and the "Post-Proline Cleaving Enzyme", which releases C-terminal dipeptide from oxytocin and arginine vasopressin, were partially purified from lamb kidney by ammonium sulfate fractionation and column chromatography. The two enzymes are shown to be separate entities with different pH profiles. The prolyl peptidase activity released the C-terminal dipeptides from oxytocin and arginine vasopressin at similar rates and was inhibited by p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, L-1-tosylamido-2-phenylethylchloromethyl ketone, Co2+, Ca2+, and Zn2+, but significantly enhanced by dithiothreitol. The prolyl peptidase preparation cleaves proline-containing peptide substrates at the Pro-X bond. The rate of cleavage is dependent on the nature of residue X and with the conditions used there is no cleavage when X equals Pro; however, cleavage occurs when X is a D isomer: [Mpr1, D-Arg8] vasopressin is inactivated at a rate similar to [Mpr1, Arg8]- and [Mpr1, Lys8] vasopressin, suggesting that the known prolonged biological action of [Mpr1, D-Arg8] vasopressin is not due to resistance to the prolyl peptidase. In all characteristics tested the lamb kidney prolyl peptidase was identical to the post-proline cleaving enzyme isolated earlier from human uterus. In vivo experiments in the cat suggested that both the glycinamide-releasing enzyme and post-proline cleaving enzyme are present and effective in inactivating neurohypophyseal hormones in the intact animal. | Partial purification and characterization of post-proline cleaving enzyme: enzymatic inactivation of neurohypophyseal hormones by kidney preparations of various species. The inactivation of the neurohypophyseal hormones arginine vasopressin and oxytocin, both 14C-labelled in the C-terminal glycine residue, by enzymes present in kidney homogenates of various species has been investigated, and some of the enzymes responsible have been partially purified and characterized. The Leu-Gly peptide bond of oxytocin is generally most effectively cleaved by kidney homogenates, although with certain species enzymic activity hydrolyzing the Pro-Leu bond is significant. Degradation of arginine vasopressin is slower than oxytocin in all species studied, and appears to occur by a different overall mechanism since cleavage of the Pro-Arg bond is more significant than hydrolysis of the Arg-Gly bond. The enzyme releasing glycinamide from oxytocin and the "Post-Proline Cleaving Enzyme", which releases C-terminal dipeptide from oxytocin and arginine vasopressin, were partially purified from lamb kidney by ammonium sulfate fractionation and column chromatography. The two enzymes are shown to be separate entities with different pH profiles. The prolyl peptidase activity released the C-terminal dipeptides from oxytocin and arginine vasopressin at similar rates and was inhibited by p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, L-1-tosylamido-2-phenylethylchloromethyl ketone, Co2+, Ca2+, and Zn2+, but significantly enhanced by dithiothreitol. The prolyl peptidase preparation cleaves proline-containing peptide substrates at the Pro-X bond. The rate of cleavage is dependent on the nature of residue X and with the conditions used there is no cleavage when X equals Pro; however, cleavage occurs when X is a D isomer: [Mpr1, D-Arg8] vasopressin is inactivated at a rate similar to [Mpr1, Arg8]- and [Mpr1, Lys8] vasopressin, suggesting that the known prolonged biological action of [Mpr1, D-Arg8] vasopressin is not due to resistance to the prolyl peptidase. In all characteristics tested the lamb kidney prolyl peptidase was identical to the post-proline cleaving enzyme isolated earlier from human uterus. In vivo experiments in the cat suggested that both the glycinamide-releasing enzyme and post-proline cleaving enzyme are present and effective in inactivating neurohypophyseal hormones in the intact animal. | 2,300 |
pubmed23n0001_1171 | Adenosine phosphyorylase activity as distinct from inosine-guanosine phosphorylase activity in Sarcoma 180 cells and rat liver. | Adenosine phosphorylase (EC 2.4.2.-) activity present in Sarcoma 180 cells grown in culture and in rat liver, is shown to be distinct from inosine-guanosine phosphorylase by several criteria: (a) treatment of Sarcoma 180 cell extract with p-chloromercuribenzoate inhibited the two activities to a different extent, (b) adenine selectively protected the adenosine phosphorylase activity of Sarcoma 180 and rat liver extract against heat inactivation, while hypoxanthine selectively protected inosine-guanosine phosphorylase activity, (c) at nearly saturating substrate concentrations and using Sarcoma 180 extract, the rates of ribosylation of a mixture of adenine + hypoxanthine or adenine + guanine, but not of hypoxanthine + guanine, were found to be almost equal to the sum of their individual rates as measured separately, (d) inosine selectively inhibited the ribosylation of hypoxanthine and guanine catalysed by Sarcoma 180 and rat liver extract while 2-chloroadenosine selectively inhibited the ribosylation of adenine and N6-furfuryladenine, (e) pH vs. activity curves were similar with hypoxanthine or guanine as the substrate but they were markedly different from the curve with adenine as the substrate. The potential role of adenosine phosphorylase activity in vivo is discussed. | Adenosine phosphyorylase activity as distinct from inosine-guanosine phosphorylase activity in Sarcoma 180 cells and rat liver. Adenosine phosphorylase (EC 2.4.2.-) activity present in Sarcoma 180 cells grown in culture and in rat liver, is shown to be distinct from inosine-guanosine phosphorylase by several criteria: (a) treatment of Sarcoma 180 cell extract with p-chloromercuribenzoate inhibited the two activities to a different extent, (b) adenine selectively protected the adenosine phosphorylase activity of Sarcoma 180 and rat liver extract against heat inactivation, while hypoxanthine selectively protected inosine-guanosine phosphorylase activity, (c) at nearly saturating substrate concentrations and using Sarcoma 180 extract, the rates of ribosylation of a mixture of adenine + hypoxanthine or adenine + guanine, but not of hypoxanthine + guanine, were found to be almost equal to the sum of their individual rates as measured separately, (d) inosine selectively inhibited the ribosylation of hypoxanthine and guanine catalysed by Sarcoma 180 and rat liver extract while 2-chloroadenosine selectively inhibited the ribosylation of adenine and N6-furfuryladenine, (e) pH vs. activity curves were similar with hypoxanthine or guanine as the substrate but they were markedly different from the curve with adenine as the substrate. The potential role of adenosine phosphorylase activity in vivo is discussed. | 2,301 |
pubmed23n0001_1172 | Purification and properties of an alpha-amylase inhibitor from wheat. | Four inhibitors of alpha-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-exchange chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60--90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract. | Purification and properties of an alpha-amylase inhibitor from wheat. Four inhibitors of alpha-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-exchange chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60--90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract. | 2,302 |
pubmed23n0001_1173 | Purification and specificity of prolyl dipeptidase from bovine kidney. | Prolyl dipeptidase (iminodipeptidase, L-prolyl-amino acid hydrolase, EC 3.4.13.8) was purified 180-fold from bovine kidney. The enzyme which was obtained in a 10% yield was completely separated from a number of known kidney peptidases including an enzyme of very similar substrate specificity, proline aminopeptidase (L-prolyl-peptide hydrolase, EC 3.4.11.5). The specific activity of the enzyme with L-prolylglycine as substrate is 1600 units of activity per mg protein. Optimum activity of the enzyme is at pH 8.75 and the molecular weight on gel filtration was estimated to be 100 000. The isoelectric point of the enzyme is pH 4.25. Studies of substrate specificity showed that the enzyme preferentially hydrolyzes dipeptides and dipeptidyl amides with L-proline or hydroxy-L-proline at the N-terminus. Longer chain substrates with N-terminal proline were not hydrolyzed. | Purification and specificity of prolyl dipeptidase from bovine kidney. Prolyl dipeptidase (iminodipeptidase, L-prolyl-amino acid hydrolase, EC 3.4.13.8) was purified 180-fold from bovine kidney. The enzyme which was obtained in a 10% yield was completely separated from a number of known kidney peptidases including an enzyme of very similar substrate specificity, proline aminopeptidase (L-prolyl-peptide hydrolase, EC 3.4.11.5). The specific activity of the enzyme with L-prolylglycine as substrate is 1600 units of activity per mg protein. Optimum activity of the enzyme is at pH 8.75 and the molecular weight on gel filtration was estimated to be 100 000. The isoelectric point of the enzyme is pH 4.25. Studies of substrate specificity showed that the enzyme preferentially hydrolyzes dipeptides and dipeptidyl amides with L-proline or hydroxy-L-proline at the N-terminus. Longer chain substrates with N-terminal proline were not hydrolyzed. | 2,303 |
pubmed23n0001_1174 | Isolation and characterization of beta-glucosidase from the cytosol of rat kidney cortex. | A procedure is described for the preparation of extensively purified beta-D-glucosidase (EC 3.2.1.21) from the cytosol fraction of rat kidney. The specific activity of the beta-glucosidase in the high speed supernatant (100 000 X g, 90 min) fraction of rat kidney homogenate is 700-fold greater than that in the same fraction from heart, skeletal muscle, lung, spleen, brain or liver. beta-Glucosidase activity co-chromatographs with beta-D-galactosidase, beta-D-fucosidase, alpha-L-arabinosidase and beta-D-xylosidase activities through the last four column steps of the purification and their specific activities are 0.26, 0.39, 0.028 and 0.017 relative to that of beta-glucosidase, respectively. The specific activity of the apparently homogeneous beta-glucosidase is 115 000 nmol of glucose released from 4-methylumbelliferyl-beta-D-glucopyranoside per mg protein per h. All five glycosidase activities possess similar pH dependency (pH optimum, 6--7) and heat lability, and co-migrate on polyacrylamide disc gels at pH 8.9 (RF, 0.67). beta-Glucosidase acitivity is inhibited competitively by glucono-(1 leads to 5)-lactone (KI, 0.61 mM) and non-competitively by a variety of sulfhydryl reagents including N-ethylmaleimide, p-chloromercuribenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid), and iodoacetic acid. Although the enzyme will release glucose from p-nitrophenyl and 4-methylumbelliferyl derivatives of beta-D-glucose, it will not hydrolyze xylosyl-O-serine, beta-D-glucocerebroside, lactose, galactosylovalbumin or trehalose. The enzyme consists of a single polypeptide chain with a molecular weight of 50 000--58 000, has a sedimentation coefficient of 4.41 S and contains a relatively large number of acidic amino acids. A study of the distribution of beta-glucosidase activity in various regions of the dissected rat kidney indicates that the enzyme is probably contained in cells of the proximal convoluted tubule. The enzyme is also present in relatively large amounts in the villus cells, but not crypt cells, of the intestine. The physiological substrate and function of the enzyme are unknown. | Isolation and characterization of beta-glucosidase from the cytosol of rat kidney cortex. A procedure is described for the preparation of extensively purified beta-D-glucosidase (EC 3.2.1.21) from the cytosol fraction of rat kidney. The specific activity of the beta-glucosidase in the high speed supernatant (100 000 X g, 90 min) fraction of rat kidney homogenate is 700-fold greater than that in the same fraction from heart, skeletal muscle, lung, spleen, brain or liver. beta-Glucosidase activity co-chromatographs with beta-D-galactosidase, beta-D-fucosidase, alpha-L-arabinosidase and beta-D-xylosidase activities through the last four column steps of the purification and their specific activities are 0.26, 0.39, 0.028 and 0.017 relative to that of beta-glucosidase, respectively. The specific activity of the apparently homogeneous beta-glucosidase is 115 000 nmol of glucose released from 4-methylumbelliferyl-beta-D-glucopyranoside per mg protein per h. All five glycosidase activities possess similar pH dependency (pH optimum, 6--7) and heat lability, and co-migrate on polyacrylamide disc gels at pH 8.9 (RF, 0.67). beta-Glucosidase acitivity is inhibited competitively by glucono-(1 leads to 5)-lactone (KI, 0.61 mM) and non-competitively by a variety of sulfhydryl reagents including N-ethylmaleimide, p-chloromercuribenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid), and iodoacetic acid. Although the enzyme will release glucose from p-nitrophenyl and 4-methylumbelliferyl derivatives of beta-D-glucose, it will not hydrolyze xylosyl-O-serine, beta-D-glucocerebroside, lactose, galactosylovalbumin or trehalose. The enzyme consists of a single polypeptide chain with a molecular weight of 50 000--58 000, has a sedimentation coefficient of 4.41 S and contains a relatively large number of acidic amino acids. A study of the distribution of beta-glucosidase activity in various regions of the dissected rat kidney indicates that the enzyme is probably contained in cells of the proximal convoluted tubule. The enzyme is also present in relatively large amounts in the villus cells, but not crypt cells, of the intestine. The physiological substrate and function of the enzyme are unknown. | 2,304 |
pubmed23n0001_1175 | Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme. | Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+. | Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme. Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+. | 2,305 |
pubmed23n0001_1176 | Partial purification and properties of a chromatin-associated phosphoprotein kinase from rat liver nuclei. | A phosphoprotein kinase (EC 2.7.1.37) KIVb, from rat liver nuclei, was purified 75-fold by phosphocellulose chromatography and gel filtration on Sephadex G-200. The enzyme, which has an apparent molecular weight of 55 000, phosphorylates casein and chromatin-bound nonhistone proteins more readily than histones or ribosomal proteins. It exhibits an absolute requirement for divalent cation with optimum activity at 15--20 mM Mg2+. Maximal kinase activity is achieved at 100 mM NaCl. The pH vs. activity curve is biphasic with optima at pH 6.5 and pH 8.0. The Km value for casein is 280 mug/ml and the Km for ATP is 6-10(-6) M. Kinase KIVb phosphorylates numerous nonhistone nuclear proteins as shown by electrophoretic analysis. The addition of kinase KIVb to reaction mixtures containing nonhistone proteins results in the phosphorylation of a spectrum of polypeptides similar to those that are phosphorylated by endogenous nuclear kinases. Nonhistone proteins bound to chromatin appear to be better substrates for KIVb than nonhistones dissociated from chromatin. A comparison of nuclear phosphoproteins phosphorylated either in the intact animal or in vitro (by the addition of kinase KIVb) indicates some differences and some similarities in the patterns of phosphorylation. | Partial purification and properties of a chromatin-associated phosphoprotein kinase from rat liver nuclei. A phosphoprotein kinase (EC 2.7.1.37) KIVb, from rat liver nuclei, was purified 75-fold by phosphocellulose chromatography and gel filtration on Sephadex G-200. The enzyme, which has an apparent molecular weight of 55 000, phosphorylates casein and chromatin-bound nonhistone proteins more readily than histones or ribosomal proteins. It exhibits an absolute requirement for divalent cation with optimum activity at 15--20 mM Mg2+. Maximal kinase activity is achieved at 100 mM NaCl. The pH vs. activity curve is biphasic with optima at pH 6.5 and pH 8.0. The Km value for casein is 280 mug/ml and the Km for ATP is 6-10(-6) M. Kinase KIVb phosphorylates numerous nonhistone nuclear proteins as shown by electrophoretic analysis. The addition of kinase KIVb to reaction mixtures containing nonhistone proteins results in the phosphorylation of a spectrum of polypeptides similar to those that are phosphorylated by endogenous nuclear kinases. Nonhistone proteins bound to chromatin appear to be better substrates for KIVb than nonhistones dissociated from chromatin. A comparison of nuclear phosphoproteins phosphorylated either in the intact animal or in vitro (by the addition of kinase KIVb) indicates some differences and some similarities in the patterns of phosphorylation. | 2,306 |
pubmed23n0001_1177 | Purification and some properties of rat liver cysteine oxidase (cysteine dioxygenase). | Cysteine oxidase (cysteine dioxygenase, EC 1.13.11.20) was purified approximately 1000-fold from rat liver. The purified enzyme (protein-B) was obtained as an inactive form, which was activated by anaerobic preincubation with L-cysteine. The active form of protein-B was inactivated during aerobic incubation to produce cysteine sulfinate. This inactivation of protein-B was protected by a distinct protein in rat liver cytoplasm, namely stabilizing protein (protein-A). The Ka and Km values for L-cysteine were 0.8-10(-3) M and 1.3-10(-3) M respectively. The enzyme was strongly inhibited by Cu+ and/or Fe2+ chelating agents but not by Cu2+ chelating agent. The optimum pH of enzyme reaction was 8.5-9.5 while that of enzyme activation was 6.8-9.5, with a broad peak. | Purification and some properties of rat liver cysteine oxidase (cysteine dioxygenase). Cysteine oxidase (cysteine dioxygenase, EC 1.13.11.20) was purified approximately 1000-fold from rat liver. The purified enzyme (protein-B) was obtained as an inactive form, which was activated by anaerobic preincubation with L-cysteine. The active form of protein-B was inactivated during aerobic incubation to produce cysteine sulfinate. This inactivation of protein-B was protected by a distinct protein in rat liver cytoplasm, namely stabilizing protein (protein-A). The Ka and Km values for L-cysteine were 0.8-10(-3) M and 1.3-10(-3) M respectively. The enzyme was strongly inhibited by Cu+ and/or Fe2+ chelating agents but not by Cu2+ chelating agent. The optimum pH of enzyme reaction was 8.5-9.5 while that of enzyme activation was 6.8-9.5, with a broad peak. | 2,307 |
pubmed23n0001_1178 | Conformations of lysine-sensitive aspartokinase. | 1. The technique of differential thermal and proteolytic inactivation has been employed as a conformational probe for the lysine-sensitive aspartokinase (EC 2.7.2.4) of Escherichia coli B. 2. L-Amino acid inhibitors of this enzyme each induce a characteristic enzyme conformation. This is evidenced by rates of thermal and proteolytic inactivation and Arrhenius activation energies for thermal inactivation which are characteristic of the amino acid present. 3. Phenylalanine and leucine binding are mutually exclusive as evidenced by competitive behavior in thermal inactivation experiments, suggesting a hydrophobic amino acid binding site with broad specificity. 4. The phenylalanine-dependent conformation and the leucine-dependent conformation differ considerably. In comparison with the native enzyme, the former is more labile to proteolysis by trypsin whereas the latter is more stable. First-order rate constants for thermal inactivation of the phenylalanine- and leucine-dependent conformations are, respectively, about one-half and one-tenth that of the native enzyme. 5. Items 3 and 4 taken together suggest that the conformations are ligand induced and do not arise via ligand stabilization of spontaneously arising conformers. | Conformations of lysine-sensitive aspartokinase. 1. The technique of differential thermal and proteolytic inactivation has been employed as a conformational probe for the lysine-sensitive aspartokinase (EC 2.7.2.4) of Escherichia coli B. 2. L-Amino acid inhibitors of this enzyme each induce a characteristic enzyme conformation. This is evidenced by rates of thermal and proteolytic inactivation and Arrhenius activation energies for thermal inactivation which are characteristic of the amino acid present. 3. Phenylalanine and leucine binding are mutually exclusive as evidenced by competitive behavior in thermal inactivation experiments, suggesting a hydrophobic amino acid binding site with broad specificity. 4. The phenylalanine-dependent conformation and the leucine-dependent conformation differ considerably. In comparison with the native enzyme, the former is more labile to proteolysis by trypsin whereas the latter is more stable. First-order rate constants for thermal inactivation of the phenylalanine- and leucine-dependent conformations are, respectively, about one-half and one-tenth that of the native enzyme. 5. Items 3 and 4 taken together suggest that the conformations are ligand induced and do not arise via ligand stabilization of spontaneously arising conformers. | 2,308 |
pubmed23n0001_1179 | The sulphatase of ox liver. XIX. On the nature of the polymeric forms of sulphatase A present in dilute solutions. | Weight-average elution volumes of sulphatase A (an arylsulphate sulphohydrolase, EC 3.1.6.1) from Sephadex G-200 have been determined as functions of protein concentration, pH, ionic strength and temperature. The results are used to calculate the apparent association equilibrium constants for tetramer formation and the associated standard-state thermodynamic parameters. While the apparent association constant decreased from 10(28) to 10(21) M-3 on increasing the pH from 4.5 to 5.6 at ionic strength 0.1, at any particular pH value studied it was relatively insensitive to temperature variation so that deltaH is close to zero and tetramer formation in solution is associated with a positive entropy change. At pH 5.0, increasing the ionic strength from 0.1 to 2 decreased the association constant by a factor of 100. Methylumbelliferone sulphate has no effect on the association of sulphatase A. The equilibrium results are used to define the degree of association of sulphatase A likely to encountered in experiments designed to elucidate its kinetic properties. In the liver lysosome, the tetramer is probably the dominant species. The monomer and tetramer of sulphatase A have similar, or identical, specific activities with nitrocatechol sulphate and 4-methylumbelliferone sulphate as substrates. With nitrocatechol sulphate, sulphatase A shows Michaelis kinetics under conditions where the monomer is the dominant species and non-Michaelis kinetics where the tetramer is dominant. There is apparently a negative cooperativity between the monomer units in the tetramer. In 2 mM sodium taurodeoxycholate and 0.035 M MnCl2, but not in 0.1 M NaCl, the tetramer shows Michaelis kinetics. This is not due to dissociation of the tetramer. The critical micellar concentration of sodium taurodeoxycholate is about 0.8 mM in both 0.1 M NaCl and 0.035 M McCl2 but the aggregation number is greater in the latter. | The sulphatase of ox liver. XIX. On the nature of the polymeric forms of sulphatase A present in dilute solutions. Weight-average elution volumes of sulphatase A (an arylsulphate sulphohydrolase, EC 3.1.6.1) from Sephadex G-200 have been determined as functions of protein concentration, pH, ionic strength and temperature. The results are used to calculate the apparent association equilibrium constants for tetramer formation and the associated standard-state thermodynamic parameters. While the apparent association constant decreased from 10(28) to 10(21) M-3 on increasing the pH from 4.5 to 5.6 at ionic strength 0.1, at any particular pH value studied it was relatively insensitive to temperature variation so that deltaH is close to zero and tetramer formation in solution is associated with a positive entropy change. At pH 5.0, increasing the ionic strength from 0.1 to 2 decreased the association constant by a factor of 100. Methylumbelliferone sulphate has no effect on the association of sulphatase A. The equilibrium results are used to define the degree of association of sulphatase A likely to encountered in experiments designed to elucidate its kinetic properties. In the liver lysosome, the tetramer is probably the dominant species. The monomer and tetramer of sulphatase A have similar, or identical, specific activities with nitrocatechol sulphate and 4-methylumbelliferone sulphate as substrates. With nitrocatechol sulphate, sulphatase A shows Michaelis kinetics under conditions where the monomer is the dominant species and non-Michaelis kinetics where the tetramer is dominant. There is apparently a negative cooperativity between the monomer units in the tetramer. In 2 mM sodium taurodeoxycholate and 0.035 M MnCl2, but not in 0.1 M NaCl, the tetramer shows Michaelis kinetics. This is not due to dissociation of the tetramer. The critical micellar concentration of sodium taurodeoxycholate is about 0.8 mM in both 0.1 M NaCl and 0.035 M McCl2 but the aggregation number is greater in the latter. | 2,309 |
pubmed23n0001_1180 | Purification and characterization of an extracellular exo-D-galacturonanase of Aspergillus niger. | A D-galacturonanase (EC 3.2.1.67) catalyzing the degradation of D-galacturonans by terminal action pattern was purified from a culture filtrate of Aspergillus niger by a procedure including the salting-out with ammonium sulfate, precipitation by ethanol, chromatography on DEAE-cellulose, and gel chromatography on Sephadex G-100. The obtained preparation was slightly contaminated by an enzymically inactive protein fraction. Maximum activity and stability of the enzyme was observed at pH 5.2. The enzyme degrades digalacturonic acid, p-nitrophenyl-alpha-D-galactopyranuronide, as well as oligogalacturonides containing at the nonreducing end 4-deoxy-L-threo-hexa-4-enopyranosyluronate. It differs from all A. niger enzymes so far described which degrade D-galaturonans by the terminal action pattern, in not clearly preferring low-molecular substrates. It is therefore classified as an exo-D-galacturonanase. | Purification and characterization of an extracellular exo-D-galacturonanase of Aspergillus niger. A D-galacturonanase (EC 3.2.1.67) catalyzing the degradation of D-galacturonans by terminal action pattern was purified from a culture filtrate of Aspergillus niger by a procedure including the salting-out with ammonium sulfate, precipitation by ethanol, chromatography on DEAE-cellulose, and gel chromatography on Sephadex G-100. The obtained preparation was slightly contaminated by an enzymically inactive protein fraction. Maximum activity and stability of the enzyme was observed at pH 5.2. The enzyme degrades digalacturonic acid, p-nitrophenyl-alpha-D-galactopyranuronide, as well as oligogalacturonides containing at the nonreducing end 4-deoxy-L-threo-hexa-4-enopyranosyluronate. It differs from all A. niger enzymes so far described which degrade D-galaturonans by the terminal action pattern, in not clearly preferring low-molecular substrates. It is therefore classified as an exo-D-galacturonanase. | 2,310 |
pubmed23n0001_1181 | The biosynthesis of multi-L-arginyl-poly(L-aspartic acid) in the filamentous cyanobacterium Anabaena cylindrica. | The cyanobacteria produce multi-L-arginyl-poly (aspartic acid), a high molecular weight (Mr=25 000-125 000) branched polypeptide consisting of a poly(aspartic acid) core with L-arginyl residues peptide bonded to each free carboxyl group of the poly(aspartic acid). An enzyme which will elongate Arg-poly(Asp) has been isolated and purified 92-fold from the filamentous cyanobacterium Anabaena cylindrica. The enzyme incorporates arginine and aspartic acid into Arg-poly(Asp) in a reaction which requires ATP, KCl, MgCl2, and a sulfhydryl reagent. The enzymatic incorporation of arginine is dependent upon the presence of L-aspartic acid but not visa versa, a finding which suggests the order of amino acid addition to the branched polypeptide-aspartic acid is added to the core followed by the attachment of an arginine branch. The elongation of Arg-poly(Asp) in-vitro is insensitive to the addition of protein synthesis inhibitors and to the addition of nucleases. These findings support the notion previosly suggested from in-vivo studies that Arg-poly(Asp) is synthesized via a non-ribosomal route and also demonstrate that amino-acetylated transfer-RNAs play no part in at least one step of the biosynthetic mechanism. | The biosynthesis of multi-L-arginyl-poly(L-aspartic acid) in the filamentous cyanobacterium Anabaena cylindrica. The cyanobacteria produce multi-L-arginyl-poly (aspartic acid), a high molecular weight (Mr=25 000-125 000) branched polypeptide consisting of a poly(aspartic acid) core with L-arginyl residues peptide bonded to each free carboxyl group of the poly(aspartic acid). An enzyme which will elongate Arg-poly(Asp) has been isolated and purified 92-fold from the filamentous cyanobacterium Anabaena cylindrica. The enzyme incorporates arginine and aspartic acid into Arg-poly(Asp) in a reaction which requires ATP, KCl, MgCl2, and a sulfhydryl reagent. The enzymatic incorporation of arginine is dependent upon the presence of L-aspartic acid but not visa versa, a finding which suggests the order of amino acid addition to the branched polypeptide-aspartic acid is added to the core followed by the attachment of an arginine branch. The elongation of Arg-poly(Asp) in-vitro is insensitive to the addition of protein synthesis inhibitors and to the addition of nucleases. These findings support the notion previosly suggested from in-vivo studies that Arg-poly(Asp) is synthesized via a non-ribosomal route and also demonstrate that amino-acetylated transfer-RNAs play no part in at least one step of the biosynthetic mechanism. | 2,311 |
pubmed23n0001_1182 | Separation of subchloroplast membrane particles by counter-current distribution. | Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity. The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique. Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks. The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicated that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae. | Separation of subchloroplast membrane particles by counter-current distribution. Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity. The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique. Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks. The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicated that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae. | 2,312 |
pubmed23n0001_1183 | Stimulation of ATP synthesis in Halobacterium halobium R1 by light-induced or artifically created proton electrochemical potential gradients across the cell membrane. | The relationship between proton movement and phosphorylation in Halo-bacterium halobium R1 has been investigated under anaerobic conditions. The light-induced changes in the bacteriorhodopsin are accompanied by proton movements across the membrane which result in pH changes in the suspending medium. The initial alkaline shift is shown to be closely paralleled by (and hence correlated with) ATP synthesis. Acidification of the medium in the presence of valinomycin, under conditions of low external potassium, brings about ATP synthesis in the dark. | Stimulation of ATP synthesis in Halobacterium halobium R1 by light-induced or artifically created proton electrochemical potential gradients across the cell membrane. The relationship between proton movement and phosphorylation in Halo-bacterium halobium R1 has been investigated under anaerobic conditions. The light-induced changes in the bacteriorhodopsin are accompanied by proton movements across the membrane which result in pH changes in the suspending medium. The initial alkaline shift is shown to be closely paralleled by (and hence correlated with) ATP synthesis. Acidification of the medium in the presence of valinomycin, under conditions of low external potassium, brings about ATP synthesis in the dark. | 2,313 |
pubmed23n0001_1184 | Mechanism of active shrinkage in mitochondria. II. Coupling between strong electrolyte fluxes. | 1. Addition of succinate to valinomycin-treated mitochondria incubated in KCL causes a large electrolyte penetration. The process depends on a steady supply of energy and involves a continuous net extrusion of protons. Rates of respiration and of electrolyte penetration proceed in a parallel manner. 2. A passive penetration of K+ salt of permeant anions occurs in respiratory-inhibited mitochondria after addition of valinomycin. Addition of succinate at the end of the passive swelling starts an active extrusion of anions and cations with restoration of the initial volume. The shrinkage is accompanied by a slow reuptake of protons. The initiation of the active shrinkage correlates with the degree of stretching of the inner membrane. The extrusion of electrolytes is inhibited by nigericin, while it is only slightly sensitive to variations of the valinomycin concentration larger than two orders of magnitude. 3. Passive swelling and active shrinkage occurs also when K+ is replaced by a large variety of organic cations. The rate of organic cation penetration is enhanced by tetraphenylboron, while the rate of electrolyte extrusion is insensitive to variation of the tetraphenylboron concentration. 4. Active shrinkage, either with K+ or organic cation salts, is inhibited by weak acids. The phosphate inhibition is removed by SH inhibitors. The active shrinkage is also inhibited by mersalyl to an extent of about 60%. 5. Three models of active shrinkage are discussed: (a) mechanoprotein, (b) electrogenic proton pump, and (c) proton-driven cation anion pump. | Mechanism of active shrinkage in mitochondria. II. Coupling between strong electrolyte fluxes. 1. Addition of succinate to valinomycin-treated mitochondria incubated in KCL causes a large electrolyte penetration. The process depends on a steady supply of energy and involves a continuous net extrusion of protons. Rates of respiration and of electrolyte penetration proceed in a parallel manner. 2. A passive penetration of K+ salt of permeant anions occurs in respiratory-inhibited mitochondria after addition of valinomycin. Addition of succinate at the end of the passive swelling starts an active extrusion of anions and cations with restoration of the initial volume. The shrinkage is accompanied by a slow reuptake of protons. The initiation of the active shrinkage correlates with the degree of stretching of the inner membrane. The extrusion of electrolytes is inhibited by nigericin, while it is only slightly sensitive to variations of the valinomycin concentration larger than two orders of magnitude. 3. Passive swelling and active shrinkage occurs also when K+ is replaced by a large variety of organic cations. The rate of organic cation penetration is enhanced by tetraphenylboron, while the rate of electrolyte extrusion is insensitive to variation of the tetraphenylboron concentration. 4. Active shrinkage, either with K+ or organic cation salts, is inhibited by weak acids. The phosphate inhibition is removed by SH inhibitors. The active shrinkage is also inhibited by mersalyl to an extent of about 60%. 5. Three models of active shrinkage are discussed: (a) mechanoprotein, (b) electrogenic proton pump, and (c) proton-driven cation anion pump. | 2,314 |
pubmed23n0001_1185 | Inhibitory effect of p-nitrothiophenol in the light on the photosystem II activity of spinach chloroplasts. | The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH'S caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiopehnol to causing the specific inhibition of Photosystem II. | Inhibitory effect of p-nitrothiophenol in the light on the photosystem II activity of spinach chloroplasts. The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH'S caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiopehnol to causing the specific inhibition of Photosystem II. | 2,315 |
pubmed23n0001_1186 | Relations between the electrical potential, pH gradient, proton flux and phosphorylation in the photosynthetic membrane. | The transmembrane electrical potential (deltaphi), the proton flux (H+), the rate of electron transport (e), the pH gradient (deltapH) and the rate of phosphorylation (ATP) were measured in chloroplasts of spinach. Photosynthesis was excited periodically with flashes of variable frequencies and intensities. A new method is described for determining the rate of electron transport and proton flux. Under conditions where the rate of electron transport and proton flux are not pH controlled the following correlations were found in the range 50 mV less than or equal to deltaphi less than or equal to 125 mV and 1.8 less than or equal to deltapH less than or equal to 2.7: (1) The pH gradient, deltapH, increases with H+ independently of Phout between 7-9. (2) The rate of phosphorylation, ATP, depends exponentially on deltapH (at constant deltaphi) and is independent of pHout between 7-9. (3) The rate of phosphorylation, ATP, depends also on deltaphi (at constant deltapH and at constant proton flux H+). (4) The proton flux via the ATPase pathway, Hp+, depends non-linearly on the ratio of the proton concentrations: Hp+ approximately (Hin+/Hout+)b, (b=2.3--2.6). The proton flux via the basal pathway, Hb+, depends linearly on the ratio of the proton concentrations: Hb+ approximately (Hin/Hout). (5) The ratio deltaH+/ATP (e/ATP, i.e. the ratio of the total proton flux, Hp+ + Hb+, and the rate of ATP formation, ATP, depends strongly on deltaphi and on deltapH. The ratio is deltaH+/ATP approximately 3 (e/ATP approximately 1.5) at deltapH 2.7 and deltaphi = 125 mV. (6) It is supposed that the reason for the dependence of deltaH+/ATP on deltaphi anddeltapH is the different functional dependence of the basal proton flux Hb+ and the phosphorylating proton flux Hp+ on deltapH and deltaphi. The calculation of deltaH+/ATP on the basis of this assumption is in fair agreement with the experimental values. Also the "threshold" effects can be explained in this way. (7) The ratio of deltaHp+/ATP, i.e. the ratio of the phosphorylating proton flux Hp+ and ATP, is deltaHp+/ATP APPROXIMATELY 2.4. | Relations between the electrical potential, pH gradient, proton flux and phosphorylation in the photosynthetic membrane. The transmembrane electrical potential (deltaphi), the proton flux (H+), the rate of electron transport (e), the pH gradient (deltapH) and the rate of phosphorylation (ATP) were measured in chloroplasts of spinach. Photosynthesis was excited periodically with flashes of variable frequencies and intensities. A new method is described for determining the rate of electron transport and proton flux. Under conditions where the rate of electron transport and proton flux are not pH controlled the following correlations were found in the range 50 mV less than or equal to deltaphi less than or equal to 125 mV and 1.8 less than or equal to deltapH less than or equal to 2.7: (1) The pH gradient, deltapH, increases with H+ independently of Phout between 7-9. (2) The rate of phosphorylation, ATP, depends exponentially on deltapH (at constant deltaphi) and is independent of pHout between 7-9. (3) The rate of phosphorylation, ATP, depends also on deltaphi (at constant deltapH and at constant proton flux H+). (4) The proton flux via the ATPase pathway, Hp+, depends non-linearly on the ratio of the proton concentrations: Hp+ approximately (Hin+/Hout+)b, (b=2.3--2.6). The proton flux via the basal pathway, Hb+, depends linearly on the ratio of the proton concentrations: Hb+ approximately (Hin/Hout). (5) The ratio deltaH+/ATP (e/ATP, i.e. the ratio of the total proton flux, Hp+ + Hb+, and the rate of ATP formation, ATP, depends strongly on deltaphi and on deltapH. The ratio is deltaH+/ATP approximately 3 (e/ATP approximately 1.5) at deltapH 2.7 and deltaphi = 125 mV. (6) It is supposed that the reason for the dependence of deltaH+/ATP on deltaphi anddeltapH is the different functional dependence of the basal proton flux Hb+ and the phosphorylating proton flux Hp+ on deltapH and deltaphi. The calculation of deltaH+/ATP on the basis of this assumption is in fair agreement with the experimental values. Also the "threshold" effects can be explained in this way. (7) The ratio of deltaHp+/ATP, i.e. the ratio of the phosphorylating proton flux Hp+ and ATP, is deltaHp+/ATP APPROXIMATELY 2.4. | 2,316 |
pubmed23n0001_1187 | A possible mechanism of the generation of singlet molecular oxygen in nadph-dependent microsomal lipid peroxidation. | A simplified system, consisting of NADPH, Fe3+-ADP, EDTA, liposomes, NADPH-cytochrome c reductase and Tris - HCl buffer (pH 6.8), has been employed in studies of the generation of singlet oxygen in NADPH-dependent microsomal lipid peroxidation. The light emitted by the system involves 1deltag type molecular oxygen identifiable by its characteristic emission spectrum and its behavior with beta-carotene. The generation of another excited species (a compound in the triplet state) could be demonstrated in this system by changes of light intensity and emission spectra which arise from photosensitizer (9,10-dibromoanthracene sulfonate, eosin, Rose-Bengal)-mediated energy transfers. Chemiluminescence in the visible region was markedly quenched by various radical trappers and by an inhibitor of NADPH-cytochrome c reductase, but not by superoxide dismutase. During the early stage of lipid peroxidation, the intensity of chemiluminescence was proportional to the square of the concentration of lipid peroxide. These characteristics suggest that singlet oxygen and a compound in the triplet state (probably a carbonyl compound) are generated by a self-reaction of lipid peroxy radicals. | A possible mechanism of the generation of singlet molecular oxygen in nadph-dependent microsomal lipid peroxidation. A simplified system, consisting of NADPH, Fe3+-ADP, EDTA, liposomes, NADPH-cytochrome c reductase and Tris - HCl buffer (pH 6.8), has been employed in studies of the generation of singlet oxygen in NADPH-dependent microsomal lipid peroxidation. The light emitted by the system involves 1deltag type molecular oxygen identifiable by its characteristic emission spectrum and its behavior with beta-carotene. The generation of another excited species (a compound in the triplet state) could be demonstrated in this system by changes of light intensity and emission spectra which arise from photosensitizer (9,10-dibromoanthracene sulfonate, eosin, Rose-Bengal)-mediated energy transfers. Chemiluminescence in the visible region was markedly quenched by various radical trappers and by an inhibitor of NADPH-cytochrome c reductase, but not by superoxide dismutase. During the early stage of lipid peroxidation, the intensity of chemiluminescence was proportional to the square of the concentration of lipid peroxide. These characteristics suggest that singlet oxygen and a compound in the triplet state (probably a carbonyl compound) are generated by a self-reaction of lipid peroxy radicals. | 2,317 |
pubmed23n0001_1188 | Primary reactions of photosystem II at low pH. I. Prompt and delayed fluorescence. | Prompt and delayed chlorophyll fluorescence have been studied in broken spinach chloroplasts at pH values down to 2.6. No direct effect of low pH on the primary charge separation in Photosystem II was observed. The irreversible inactivation of a secondary electron donor in a narrow pH range around pH 4.5 was demonstrated. At lower pH values the photooxidized form of a more primary electron donor, revealed by its efficient fluorescence quenching, was reduced with a half time of about 200 mus, 25% by another electron donor and 75% by back reaction with the reduced acceptor. The electron donation had a half time of 800 mus and was practically irreversible. The back reaction had a pH dependent half time: about 270 mus at pH 4 and increasing towards lower pH. The competition of both reactions resulted in a net efficiency of the charge separation at pH 4 of 25%, increasing towards lower pH. | Primary reactions of photosystem II at low pH. I. Prompt and delayed fluorescence. Prompt and delayed chlorophyll fluorescence have been studied in broken spinach chloroplasts at pH values down to 2.6. No direct effect of low pH on the primary charge separation in Photosystem II was observed. The irreversible inactivation of a secondary electron donor in a narrow pH range around pH 4.5 was demonstrated. At lower pH values the photooxidized form of a more primary electron donor, revealed by its efficient fluorescence quenching, was reduced with a half time of about 200 mus, 25% by another electron donor and 75% by back reaction with the reduced acceptor. The electron donation had a half time of 800 mus and was practically irreversible. The back reaction had a pH dependent half time: about 270 mus at pH 4 and increasing towards lower pH. The competition of both reactions resulted in a net efficiency of the charge separation at pH 4 of 25%, increasing towards lower pH. | 2,318 |
pubmed23n0001_1189 | Immunological similarity between NADH-cytochrome b5 reductase of erythrocytes and liver microsomes. | In a number of animal species soluble NADH-cytochrome b5 reductase of erythrocytes was compared with membrane-bound NADH-cytochrome b5 reductase of liver microsomes by using an antibody to purified NADH-cytochrome b5 reductase from rat liver microsomes. The results obtained indicated clearly that they are immunologically very similar to each other. The data with erythrocyte ghosts suggested that cytochrome b5 and NADH-cytochrome b5 reductase are also present in the ghost. | Immunological similarity between NADH-cytochrome b5 reductase of erythrocytes and liver microsomes. In a number of animal species soluble NADH-cytochrome b5 reductase of erythrocytes was compared with membrane-bound NADH-cytochrome b5 reductase of liver microsomes by using an antibody to purified NADH-cytochrome b5 reductase from rat liver microsomes. The results obtained indicated clearly that they are immunologically very similar to each other. The data with erythrocyte ghosts suggested that cytochrome b5 and NADH-cytochrome b5 reductase are also present in the ghost. | 2,319 |
pubmed23n0001_1190 | Effect of NADP on light-induced cytochrome changes in membrane fragments from a blue-green alga. | The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water. | Effect of NADP on light-induced cytochrome changes in membrane fragments from a blue-green alga. The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water. | 2,320 |
pubmed23n0001_1191 | Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing. | 1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed. | Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing. 1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed. | 2,321 |
pubmed23n0001_1192 | Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium. | We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic (t1/2 of less than 5 mus and approximately 50 mus). We belive that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll. We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below -450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD+ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae. | Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium. We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic (t1/2 of less than 5 mus and approximately 50 mus). We belive that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll. We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below -450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD+ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae. | 2,322 |
pubmed23n0001_1193 | Reaction of uracil and thymine derivatives with sodium bisulfite. Studies on the mechanism and reduction of the adduct. | The rates and equilibria for the addition of sodium bisulfite to uracil, thymine, and their nucleosides have been studied for the pH range 3-9.5. The rate of addition for uracil is proportional to the concentration of sulfite ion and unionized uracil. The equilibrium constant (25 degrees C) for the reaction is (1.0 +/- 0.15) X 10(3) 1 - mol-1 for uracil and 0.62 +/- 0.03 1- mol-1 for thymine. A pH of 6-7, with a high bisulfite concentration is suggested for biochemical applications of the uracil reaction. The uracil reaction, which proceeds readily under physiological conditions and has a high equilibrium constant, may be a contributing cause of the biochemical effects of bisulfite and sulfur dioxide. Additional evidence on the structure of the thymine-bisulfite adduct has been obtained by nuclear magnetic resonance spectroscopy. This spectrum supports the assignment of structure as dihydrothymine-6-sulfonate. The uracil-bisulfite adduct is reduced by sodium borohydride to sodium 3-ureido-propanol-2-sulfonate. This reaction is suggested for the chemical modification of nucleic acids. | Reaction of uracil and thymine derivatives with sodium bisulfite. Studies on the mechanism and reduction of the adduct. The rates and equilibria for the addition of sodium bisulfite to uracil, thymine, and their nucleosides have been studied for the pH range 3-9.5. The rate of addition for uracil is proportional to the concentration of sulfite ion and unionized uracil. The equilibrium constant (25 degrees C) for the reaction is (1.0 +/- 0.15) X 10(3) 1 - mol-1 for uracil and 0.62 +/- 0.03 1- mol-1 for thymine. A pH of 6-7, with a high bisulfite concentration is suggested for biochemical applications of the uracil reaction. The uracil reaction, which proceeds readily under physiological conditions and has a high equilibrium constant, may be a contributing cause of the biochemical effects of bisulfite and sulfur dioxide. Additional evidence on the structure of the thymine-bisulfite adduct has been obtained by nuclear magnetic resonance spectroscopy. This spectrum supports the assignment of structure as dihydrothymine-6-sulfonate. The uracil-bisulfite adduct is reduced by sodium borohydride to sodium 3-ureido-propanol-2-sulfonate. This reaction is suggested for the chemical modification of nucleic acids. | 2,323 |
pubmed23n0001_1194 | Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation. | Physicochemical properties of mixtures of spectrin and actin extracted from human erythrocyte ghosts have been correlated with ultrastructural changes observed in freeze-fractured erythrocyte membranes. (1) Extracted mixtures of spectrin and actin have a very low solubility (less than 30 mug/ml) near their isoelectric point, pH 4.8. These mixtures are also precipitated by low concentrations of Ca2+, Mg2+, polylysine or basic proteins. (2) All conditions which precipitate extracts of spectrin and actin also induce aggregation of the intramembrane particles in spectrin-depleted erythrocyte ghosts. Precipitation of the residual spectrin molecules into small patches on the cytoplasmic surface of the ghost membrane is thought to be the cause of particle aggregations, implying an association between the spectrin molecules and the intramembrane particles. (3) When fresh ghosts are exposed to conditions which precipitate extracts of spectrin and actin, only limited particle aggregation occurs. Instead, the contraction of the intact spectrin meshwork induced by the precipitation conditions compresses the lipid bilayer of the membrane, causing it to bleb off particle-free, protein-free vesicles. (4) The absence of protein in these lipid vesicles implies that all the proteins of the erythrocyte membrane are immobilized by association with either the spectrin meshwork or the intramembrane particles. | Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation. Physicochemical properties of mixtures of spectrin and actin extracted from human erythrocyte ghosts have been correlated with ultrastructural changes observed in freeze-fractured erythrocyte membranes. (1) Extracted mixtures of spectrin and actin have a very low solubility (less than 30 mug/ml) near their isoelectric point, pH 4.8. These mixtures are also precipitated by low concentrations of Ca2+, Mg2+, polylysine or basic proteins. (2) All conditions which precipitate extracts of spectrin and actin also induce aggregation of the intramembrane particles in spectrin-depleted erythrocyte ghosts. Precipitation of the residual spectrin molecules into small patches on the cytoplasmic surface of the ghost membrane is thought to be the cause of particle aggregations, implying an association between the spectrin molecules and the intramembrane particles. (3) When fresh ghosts are exposed to conditions which precipitate extracts of spectrin and actin, only limited particle aggregation occurs. Instead, the contraction of the intact spectrin meshwork induced by the precipitation conditions compresses the lipid bilayer of the membrane, causing it to bleb off particle-free, protein-free vesicles. (4) The absence of protein in these lipid vesicles implies that all the proteins of the erythrocyte membrane are immobilized by association with either the spectrin meshwork or the intramembrane particles. | 2,324 |
pubmed23n0001_1195 | The rate of calcium uptake into sarcoplasmic reticulum of cardiac muscle and skeletal muscle. Effects of cyclic AMP-dependent protein kinase and phosphorylase b kinase. | Calcium transport into sarcoplasmic reticulum fragments isolated from dog cardiac and mixed skeletal muscle (quadriceps) and from mixed fast (tibialis), pure fast (caudofemoralis) and pure slow (soleus) skeletal muscles from the cat was studied. Cyclic AMP-dependent protein kinase and phosphorylase b kinase stimulated the rate of calcium transport although some variability was observed. A specific protein kinase inhibitor prevented the effect of protein kinase but not of phosphorylase b kinase. The addition of cyclic AMP to the sarcoplasmic reticulum preparations in the absence of protein kinase had only a slight stimulatory effect despite the presence of endogenous protein kinase. Cyclic AMP-dependent protein kinase catalyzed the phosphorylation of several components present in the sarcoplasmic reticulum fragments; a 19000 to 21 000 dalton peak was phosphorylated with high specific activity in sarcoplasmic reticulum preparations isolated from heart and from slow skeletal muscle, but not from fast skeletal muscle. Phosphorylase b kinase phosphorylated a peak of molecular weight 95000 in all of the preparations. Cyclic AMP-dependent protein kinase-stimulated phosphorylation was optimum at pH 6.8; phosphorylase b kinase phosphorylation had a biphasic curve in cardiac and slow skeletal muscle with optima at pH 6.8 and 8.0. The addition of exogenous phosphorylase b kinase or protein kinase increased the endogenous level of phosphorylation 25-100%. All sarcoplasmic reticulum preparations contained varying amounts of adenylate cyclase, phosphorylase b and a (b:a = 30.1), "debrancher" enzyme and glycogen (0.3 mg/mg protein), as well as varying amounts of protein kinase and phosphorylase b kinase which were responsible for a significant endogenous phosphorylation. Thus, the two phosphorylating enzymes stimulated calcium uptake in the sarcoplasmic reticulum of a variety of muscles possessing different physiologic characteristics and different responses to drugs. In addition, the phosphorylation catalyzed by these enzymes occurred at two different protein moieties which make physiologic interpretation of the role of phosphorylation difficult. While the role phosphorylation in these mechanisms is complex, the presence of a glycogenolytic enzyme system may be an important link in this phenomenon. The sarcoplasmic reticulum represents a new substrate for phosphorylase b kinase. | The rate of calcium uptake into sarcoplasmic reticulum of cardiac muscle and skeletal muscle. Effects of cyclic AMP-dependent protein kinase and phosphorylase b kinase. Calcium transport into sarcoplasmic reticulum fragments isolated from dog cardiac and mixed skeletal muscle (quadriceps) and from mixed fast (tibialis), pure fast (caudofemoralis) and pure slow (soleus) skeletal muscles from the cat was studied. Cyclic AMP-dependent protein kinase and phosphorylase b kinase stimulated the rate of calcium transport although some variability was observed. A specific protein kinase inhibitor prevented the effect of protein kinase but not of phosphorylase b kinase. The addition of cyclic AMP to the sarcoplasmic reticulum preparations in the absence of protein kinase had only a slight stimulatory effect despite the presence of endogenous protein kinase. Cyclic AMP-dependent protein kinase catalyzed the phosphorylation of several components present in the sarcoplasmic reticulum fragments; a 19000 to 21 000 dalton peak was phosphorylated with high specific activity in sarcoplasmic reticulum preparations isolated from heart and from slow skeletal muscle, but not from fast skeletal muscle. Phosphorylase b kinase phosphorylated a peak of molecular weight 95000 in all of the preparations. Cyclic AMP-dependent protein kinase-stimulated phosphorylation was optimum at pH 6.8; phosphorylase b kinase phosphorylation had a biphasic curve in cardiac and slow skeletal muscle with optima at pH 6.8 and 8.0. The addition of exogenous phosphorylase b kinase or protein kinase increased the endogenous level of phosphorylation 25-100%. All sarcoplasmic reticulum preparations contained varying amounts of adenylate cyclase, phosphorylase b and a (b:a = 30.1), "debrancher" enzyme and glycogen (0.3 mg/mg protein), as well as varying amounts of protein kinase and phosphorylase b kinase which were responsible for a significant endogenous phosphorylation. Thus, the two phosphorylating enzymes stimulated calcium uptake in the sarcoplasmic reticulum of a variety of muscles possessing different physiologic characteristics and different responses to drugs. In addition, the phosphorylation catalyzed by these enzymes occurred at two different protein moieties which make physiologic interpretation of the role of phosphorylation difficult. While the role phosphorylation in these mechanisms is complex, the presence of a glycogenolytic enzyme system may be an important link in this phenomenon. The sarcoplasmic reticulum represents a new substrate for phosphorylase b kinase. | 2,325 |
pubmed23n0001_1196 | [Microcolorimetric study of the association of trypsin with a pancreatic inhibitor]. | Enthalpy of the association of trypsin with pancreatic inhibitor from bovine pancreas at 25 degrees C as a function of pH and ionic strength is estimated. The dependence of the enthalpy on pH is of an extremal character with a minimum at pH 7.6 (delta H degrees =-10.3 ccal/mole). The increase of ionic strength with the addition of LiCl, KCl and CsCl at pH 7.6 leads to be increase of delta H degrees. Possible mechanisms of enthlpy changes depending on medium conditions are considered. | [Microcolorimetric study of the association of trypsin with a pancreatic inhibitor]. Enthalpy of the association of trypsin with pancreatic inhibitor from bovine pancreas at 25 degrees C as a function of pH and ionic strength is estimated. The dependence of the enthalpy on pH is of an extremal character with a minimum at pH 7.6 (delta H degrees =-10.3 ccal/mole). The increase of ionic strength with the addition of LiCl, KCl and CsCl at pH 7.6 leads to be increase of delta H degrees. Possible mechanisms of enthlpy changes depending on medium conditions are considered. | 2,326 |
pubmed23n0001_1197 | [Partial purification and properties of protease from Torula thermophila]. | 11-Fold purified protease preparation is isolated from cultural medium of Torula thermophila UzPT-1 by means of ammonium sulphate precipitation and gel chromatography through Sephadex G-100. Disc polyacrylamide gel electrophoresis revealed two portease components, one of them possessing proteolytic activity. pH interval for protease activity was found to be 3.5-12, the maximal activity was observed at pH 8.5-11, the highest enzyme resistance--at pH 6-8. The enzyme almost completely preserved its activity for 1 hour in distilled water at 60 degrees C. The temperature maximum of the enzyme activity was 70 degrees at pH 8. The enzyme may be referred to proteases of serine nature, because it is completely inactivated with diisopropylphosphofluoridate, but it retains the activity in the presence of chelating agents (EDTA, o-phenantroline, ditizone) and inhibitors of SH-groups (sodium p-chloromercuriumbenzoate, iodoacetic acid). The enzyme was not inactivated with phenylmethylsulphonylfluoride and the trypsin inhibitor from soybean. The protease studied most efficiently hydrolyzed caseine and hemoglobin, in a less degree--human serum albumin and fibrinogen and almost did not attack egg albumin. The enzyme undergoes association-dissociation under pH change during gel filtration through Sephadex. | [Partial purification and properties of protease from Torula thermophila]. 11-Fold purified protease preparation is isolated from cultural medium of Torula thermophila UzPT-1 by means of ammonium sulphate precipitation and gel chromatography through Sephadex G-100. Disc polyacrylamide gel electrophoresis revealed two portease components, one of them possessing proteolytic activity. pH interval for protease activity was found to be 3.5-12, the maximal activity was observed at pH 8.5-11, the highest enzyme resistance--at pH 6-8. The enzyme almost completely preserved its activity for 1 hour in distilled water at 60 degrees C. The temperature maximum of the enzyme activity was 70 degrees at pH 8. The enzyme may be referred to proteases of serine nature, because it is completely inactivated with diisopropylphosphofluoridate, but it retains the activity in the presence of chelating agents (EDTA, o-phenantroline, ditizone) and inhibitors of SH-groups (sodium p-chloromercuriumbenzoate, iodoacetic acid). The enzyme was not inactivated with phenylmethylsulphonylfluoride and the trypsin inhibitor from soybean. The protease studied most efficiently hydrolyzed caseine and hemoglobin, in a less degree--human serum albumin and fibrinogen and almost did not attack egg albumin. The enzyme undergoes association-dissociation under pH change during gel filtration through Sephadex. | 2,327 |
pubmed23n0001_1198 | [Various properties of the creatine transport system and the location of creatine kinase in skeletal muscle mitochondria]. | Water and creatine contents were studied in rat skeletal muscle mitochondria after their 5 min. incubation in creatine solutions, pH 7.2 or 8.4. The content of water and creatine in mitochondria was found to be higher at pH 8.4, than at pH 72, the creatine content correlated with the water content. Structural creatine analogues, containing aminogroups with pKa greater than or equal to 9.5 or carboxyl groups, inhibited the infusion of creatine into mitochondria more strongly than substances having aminogroups with pKa less than 5. The penetrating form is creatine amphiion; the effect of pH on the permeability is probably due to the activation of the creatine transmitter. Rat skeletal muscle mitochondria contain creatine kinase at both sides of the inner membrane. This conclusion is based on the fact that under conditions, supplying the direct course of the creatine kinase reaction (the incubation medium contains Ca2+ and creatine; pH 7.8), ADP produces the stimulation of mitochondrial respiration up to the oxygen exhausting in a polarographic unit. Similarly, ADP irreversibly stimulates mitochondrial respiration in the presence of 1 mM EDTA, if EDTA and ADP are added after the preincubation of mitochondria in creatine-containing medium and after accumulating small amounts of Ca2+ by mitochondria. | [Various properties of the creatine transport system and the location of creatine kinase in skeletal muscle mitochondria]. Water and creatine contents were studied in rat skeletal muscle mitochondria after their 5 min. incubation in creatine solutions, pH 7.2 or 8.4. The content of water and creatine in mitochondria was found to be higher at pH 8.4, than at pH 72, the creatine content correlated with the water content. Structural creatine analogues, containing aminogroups with pKa greater than or equal to 9.5 or carboxyl groups, inhibited the infusion of creatine into mitochondria more strongly than substances having aminogroups with pKa less than 5. The penetrating form is creatine amphiion; the effect of pH on the permeability is probably due to the activation of the creatine transmitter. Rat skeletal muscle mitochondria contain creatine kinase at both sides of the inner membrane. This conclusion is based on the fact that under conditions, supplying the direct course of the creatine kinase reaction (the incubation medium contains Ca2+ and creatine; pH 7.8), ADP produces the stimulation of mitochondrial respiration up to the oxygen exhausting in a polarographic unit. Similarly, ADP irreversibly stimulates mitochondrial respiration in the presence of 1 mM EDTA, if EDTA and ADP are added after the preincubation of mitochondria in creatine-containing medium and after accumulating small amounts of Ca2+ by mitochondria. | 2,328 |
pubmed23n0001_1199 | [Isolation and properties of a homogeneous L-asparaginase preparation from Pseudomonas fluorescens AG]. | Highly purified L-asparaginase having a specific activity of 500+/- +/-40 IU./mg protein is isolated from Pseudomonas fluorescens AG cells. The purification procedure includes isopropanol fractionation, gel filtration through Sephadex G-100, chromatography on hydroxylapatite and DEAE-cellulose columns. The asparaginase preparation is homogenous on the basis of polyacrylamide gel electrophoresis data. The pH optimum is found to be 8.0-9.0, isoelectric point and molecular weight are 4.5+/-0.05 and 70,000+/-5,000 respectively, Km for L-asparagine being-4.1-10(-4)M. The enzyme does not hydrolyse L-glutamine. The hydrolysis rate of D-glutamine is less than 1% of the deamydation rate of L-isomer. p-Chloro-mercurium benzoate at a concentration of 10(-4) M completely inhibits the asparaginase activity. Asparaginase from Ps. fluorescens AG possesses and antileucosic activity, inhibiting 3H-thymidine incorporation into DNA of Berkit lymphoma cells. | [Isolation and properties of a homogeneous L-asparaginase preparation from Pseudomonas fluorescens AG]. Highly purified L-asparaginase having a specific activity of 500+/- +/-40 IU./mg protein is isolated from Pseudomonas fluorescens AG cells. The purification procedure includes isopropanol fractionation, gel filtration through Sephadex G-100, chromatography on hydroxylapatite and DEAE-cellulose columns. The asparaginase preparation is homogenous on the basis of polyacrylamide gel electrophoresis data. The pH optimum is found to be 8.0-9.0, isoelectric point and molecular weight are 4.5+/-0.05 and 70,000+/-5,000 respectively, Km for L-asparagine being-4.1-10(-4)M. The enzyme does not hydrolyse L-glutamine. The hydrolysis rate of D-glutamine is less than 1% of the deamydation rate of L-isomer. p-Chloro-mercurium benzoate at a concentration of 10(-4) M completely inhibits the asparaginase activity. Asparaginase from Ps. fluorescens AG possesses and antileucosic activity, inhibiting 3H-thymidine incorporation into DNA of Berkit lymphoma cells. | 2,329 |
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