
Furthermore, 35S-labelled sporangiomycin binds specifically to 50 S particles.
Step-wise release of groups of ribosomal proteins by treatment with increasing concentrations of LiCl has shown that a specific fraction (the 1.3~1.7 M LiCl split proteins)
is essential for antibiotic binding to the 50S particle. It is hypothesized that sporangiomycin inhibits protein synthesis by binding to a ribosomal multimolecular site of the utmost importance in the process of peptidechain elongation.
Sporangiomycin is a peptide antibiotic isolated from the actinomyces Planomonospora parontosporal). On the basis of its chemical properties sporangiomycin is related to the thiostrepton group antibiotics, which includes thiostrepton, siomycin and thiopeptin1'^; however its chemical structure has not been conclusively determined.
The thiostrepton group antibiotics have been shown to interact specifically with the 50S ribosomal subunit of Escherichia coli thus preventing the binding of aminoacyl-tRNA and of EF-G factor3>4>5>6>7>8>9). The results presented in this paper show that sporangiomycin inhibits protein synthesis by a similar mechanism and that S5S-labelled sporangiomycin binds specifically to the 50S subunit in a 1 :1 molar ratio. Furthermore, step-wise detachment of ribosomal proteins from the 50S subunit with increasing concentrations of LiCl has shown that the split proteins contained in the 1.3~1.7 M LiCl fraction are essential for the binding of the antibiotic. Magni) ribosomes and polymerizing enzymes were prepared as described13). uC-PhenylalaninetRNA (14C-phe-tRNA) was prepared according to Kaji, Kaji and NovelliU) Yeasts and other fungi are not affected15. Fig. 1 shows that the addition of sporangiomycin to an actively growing culture of B. subtilis immediately stops protein synthesis, but not RNAor DNAsynthesis. These results suggest that the primary site of action of the antibiotic ought to be searched for in the machinery for protein synthesis.
Effect of Sporangiomycin on the Reactions for Protein Synthesis in vitro Sporangiomycin efficiently inhibits the reactions for peptide chain-elongation when added to cell-free systems prepared from B. subtilis and E. coli, while it has no effect on the systems prepared from the yeast S. cerevisiae (Fig. 2) . These results suggest that sporangiomycin is a specific inhibitor of the procaryotictype mechanism for protein synthesis, the "in vivo" resistance of gram-negative bacteria being possibly due to impermeability of the cell envelopes or to the presence of an antibiotic degrading system. Table 1 shows that the inhibition of the reactions for peptide chain-elongation by sporangiomycin can be overcome by an excess of 50S subunits, but not of 30S or supernatant enzymes. Thus the action of sporangiomycin appears to be related to the 50S subunit. Table 2 shows that E. coli and B. subtilis ribosomes pretreated with sporangiomycin are inactive in protein synthesis. A possible explanation, confirmed by the experiments described below, is that ribosomes bind the antibiotic and that the complex is stable under the experimental conditions used in the assays.
An analysis of the inhibitory effect of sporangiomycin on the reaction for protein synthesis in vitro has shown that the antibiotic interferes strongly with EF-T for 14C-uracil and 14C-phenylalanine incorporation, were grown in 50ml of the Von Borstell M40 medium. When cell concentration reached 108 cell/ml, 5 fxg/ml of 14C-phenylalanine (sp. act. 0. 017 fiC/pmole) or of 2-i4C-uracil (sp. act. 0.017fiC/ptmole) or of 2-14C-thymine (sp. act. 0.05ftC/pm.ole' ) were added.
Sporangiomycin (0. 35 fig/ml) was added after 10 minutes to 25 ml aliquots of the cultures. Two-ml samples were withdrawn at intervals, diluted with 2ml of cold 10%-TCA and the radioactivity of the insoluble fraction determined in a thin window counter (25 % efficiency). The composition of reaction mixtures and assay conditions were as described in Fig. 2 Ribosomes were then pelleted by ultracentrifugation and resuspended in the same buffer. Treated and/or untreated ribosomes were used at the concentration of 0.40 mg/ml, in absence of antibiotic as described in Fig. 2 . The mixture containing untreated ribosomes incorporated respectively 4.5 and 5. 3^moles phenylalanine in the systems containing untreated ribosomes from E. coli or B.subtilis. dependent binding of phe-tRNA to E. coli ribosomes (Fig. 3B) Binding of 85S-Sporangiomycin to Ribosomes A study of the mechanism by which sporangiomycin interferes both with EF-T and EF-G functions on the bacterial ribosome hopefully will shed some light on a ribosomal site of great functional importance. As a first step toward the definition of this problem it is important to determine if sporangiomycin specifically binds to ribosomes and which ribosomal component(s) is involved in such binding. To this purpose we have undertaken a study of the interactions between 35S-labelled sporangiomycin and ribosomes or ribosomal derived particles. Fig. 4 shows that 85S-sporangiomycin binds to the 50S subunit (but not to the 30S) of the bacterial ribosome (E. coli and B. subtilis), while it does not bind to eukaryotic type ribosomes (S. cerevisiae). Furthermore from these and similar experiments under saturating concentrations Assay conditions were as described under "Materials and Methods". The 100/d-reaction mixture for binding of fMet-tRNA and synthesis of fMet-puromycin contained:
100mMTris-HCl (pH 7.2), 5mMMg
acetate, 50mM KC1, 5mM /3 mercaptoethanol, 0.5mM GTP, 0.8 absorbance units/ml ApUpG, 422^moles/ml f-* 4C-Met-tRNA (specific activity 233 jumole), 0.9 mg/ml ribosomes, 0.41 mg/ml initiation factors. Incubation was for 15minutes at 24°C. The 100^1 reaction mixture for the assay of binding of phe-tRNA contained : 50mMTris-HCl (pH 7.8); 7mMor 20mM Mg acetate ; 160mM NH4C1 ; 2mMdithiothreitol ; 0.2 mMGTP ; 50 fig/ml polyuridilic acid ; 100 />moles/ml 14C-phenylalaninetRNA (specific activity 265 juC/jumole); 1.25 mg/ml ribosomes ; 90 jug/ml of E. coli EF-T factor were present when enzymatic binding was assayed. On the other hand, no binding site exists on the ribosome of S. cerevisiae, which has been shown to be insensitive to sporangiomycin (Fig.   2 ).
An investigation on the stability of the 35S-sporangiomycin-ribosomecomplexwas performed as described in with ribosomes (10mg/ml) prepared from E.colt, B.subtilis or S. cerevisiae in the "reconstitution buffer" containing : 10 mM Tris-HCl (pH 7.8), 20mM MgCl2 300mM KC1 and 6mM/3 mercaptoethanol. Total volume was 500jul. The mixtures were then placed on the top of a 28 ml 5-20% linear sucrose gradient containing 10mM Tris-HCl (pH 7.8), 50mM NH4C1, 6 mM /3-mercaptoethanol and 0.5niM Mgacetate (5.0mM in experiment A) andcentrifuged at21.000rpmfor 16 hours at 4°C in an SW25 Spinco rotor. One ml fractions were collected with an ISCO Table 3 . A convenient analytical method was provided by the observation that sporangiomycin is soluble in 10 %trichloro-acetic acid, while the sporangiomycinribosome complex is precipitated in the expected 1 : 1 molar ratio.
The results showed that the preformed complex was unstable to treatment with urea and Na dodecyl sulphate (SDS), known to affect largely hydrophobic bonds, while it was not affected by salts which interact with ionic bonds.
Binding of 35S-Sporangiomycin to
Ribosome-Derived Core Particles
In order to attempt an identification of the ribosomal component(s) responsible for the binding of sporangiomycin we have studied the binding capacity of particles derived from the 50S subunit by step-wise release of specific groups of ribosomal Thirty mg of E. coli ribosomes were incubated with a two-fold molar excess of 35S-sporangiomycin in a total volume of 7.5ml as described in the legendtoFig.
4. Aliquots of0.5mlwere treated for 1hour at 4°C with the reported reagent in a buffer containing 10mMTris-HCl (pH7.8), 50mM NH4C1, 5mMMg acetate and 6mM/3 mercaptoethanol.
Final volume was 5 ml. Incubation was stopped by adding an equal volume of 20 % TCA. 35S-Sporangiomycin bound to ribosomes was recovered on cellulosefilters while the unbound antibiotic, which is soluble in 10 % TCA, was eliminated by repeated washes. Control experiments bound sporangiomycin in a 1 : 1 molar ratio. 30mMTris-HCl(pH 7.8) and 100mMMg acetate in a final volume of 1ml. After incubation for 16hours at 4°C core particles were recovered by centrifuging 6 michael25@example.com^-.
The pellet was assayed for its capacity to bind 35S-sporangiomycin as described in the legend to Fig. 4 . were resuspended in the "reconstitution buffer" (as described in Fig. 4 ) and the 0~1.3 M LiCl and 1.3-1.7 m LiCl split proteins were dialyzed for 20 hours against the same buffer.
50S Ribosomal particles were partially or totally reconstituted by combining the 1.7 M LiCl core particles with both split protein fractions, or with the 1.3~1.7 m LiCl fraction, as described by Nomura and Erdmann205. The particles were then assayed for their capacity to bind 35S-sporangiomycin as described in the legend to Fig. 4. 35 different proteins present on the subunit appear to play a role in the binding of the antibiotic.
However, further work is needed in order to identify the protein(s) directly involved and to determine its function in protein synthesis.
The thiostrepton group antibiotics, to which sporangiorhycin is closely related, have been shown to interfere both with EF-T and EF-G activity. 

{'paper_id': '26206849', 'title': 'INTERACTION OF SPORANGIOMYCIN WITH THE BACTERIAL RIBOSOME', 's2_url': 'https://api.semanticscholar.org/CorpusID:26206849', 'source': 'S2ORC'}