Immunological probe of estrogen biosynthesis. Evidence for the 2 beta-hydroxylative pathway in aromatization of androgens.
The terminal hydroxylation in placental estrogen biosynthesis from androgens is at the 2 beta position. The 2 beta-hydroxy-19-oxoandrogen derivative collapses nonenzymatically to estrogen and is therefore the proximate precursor of the female hormone. To establish the role of this pathway in biological aromatization, an immunological approach was employed in which an antibody was obtained which recognizes 2 beta-hydroxy-19-oxygenated androgens but not intermediates oxygenated at C-19 only. Binding of the 2 beta-hydroxy-19-oxo intermediate by the antibody stabilizes it so that its nonenzymatic transformation to estrogen is delayed and results in slower estrogen formation. When placental microsomes were incubated with [1,2-3H]androstenedione in the presence of the antibody antiserum, a 50% decrease in [3H]estradiol formation and 3H2O release was observed when compared with identical incubations containing normal rabbit serum alone. This inhibition is blocked when the antibody is inactivated by presaturation with 2 beta, 19-dihydroxyandrostenedione. Precipitation of immunoglobulins from the incubations followed by heating liberated the 2 beta-hydroxy-19-oxo intermediate (30%) from the antibody, and resulted in its nonenzymatic collapse to estrogen with concomitant release of 3H2O. Control normal rabbit serum or blocked antibody incubations did not show a similar increase in [3H]estradiol or 3H2O yields in the precipitate. Heat treatment (90 degrees C) of the antibody but not normal rabbit serum incubations resulted in a similar increase in [3H]estradiol and 3H2O yields. These results are consistent with the hypothesis that the final and rate-determining hydroxylation in aromatization of androgens is at the 2 beta position and that this pathway is the dominant, if not the sole, route of estrogen biosynthesis by placental aromatase. The antibody probe also permits the characterization of aromatization mechanisms in tissues other than the placenta.