Placental Microsomes

In order to study the initial as well as the final steps in the aromatization of androgens to estrogens, highspecific activity [ 19-CSHs]androstenedione and testosterone were synthesized. Incubations of [ 19-CSHs]androstenedione with human placental microsomes resulted in the generation of [‘Hlwater, as a result of the dual hydroxylation at (2-19, and [’Hlformic acid reflecting final aromatization. After an initial lag in the production of [’Hlforrnic acid, the two radiolabeled products were formed linearly with time at a ratio of 2 to 1 under subsaturating conditions and 2.2 to 1 when saturating levels of substrate were present. Incubation of a mixture of [19-CSHs]and [4-14C]androstenedione with human placental microsomes yielded 19-hydroxyand 19-oxoandrostenedione, respectively, products of one and two hydroxylations at C-19. The isotope ratios of these derivatives revealed the presence of a tritium isotope effect in the first but not in the second hydroxylation at that site. When [ 19C3Hz]and [4-’4C]19-hydr~xyandr~stenedione were used as the substrate, the isotope ratio of the isolated 19-oxoandrostenedione showed no evidence of any isotope effect in its formation. Thus, the second hydroxylation at C-19 exhibits no isotope effect irrespective of whether androstenedione or 19-hydroxyandrostenedione are the substrates, and therefore, a concerted process and catalytic commitment are not responsible for the difference in isotope effects between the first and second (2-19 hydroxylation by the placental aromatase complex. Radiometric kinetic analysis employing [ 19-CSH& and [ 1/3,2/3-SHlandrostenedione as the comparative substrates provided evidence that the isotope effect is exerted solely through the V,,, component of the reaction. The distinction between the successive hydroxylations at C-19 in the aromatization sequence suggests, but does not prove, that different mechanisms, and hence different catalytic sites, may be involved in these steps.