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  • Title: Radiometric analysis of oxidative reactions in aromatization by placental microsomes. Presence of differential isotope effects.
    Author: Miyairi S, Fishman J.
    Journal: J Biol Chem; 1985 Jan 10; 260(1):320-5. PubMed ID: 3880740.
    Abstract:
    In order to study the initial as well as the final steps in the aromatization of androgens to estrogens, high-specific activity [19-C3H3]androstenedione and testosterone were synthesized. Incubations of [19-C3H3]androstenedione with human placental microsomes resulted in the generation of [3H]water, as a result of the dual hydroxylation at C-19, and [3H]formic acid reflecting final aromatization. After an initial lag in the production of [3H]formic 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-C3H3]- and [4-14C]androstenedione with human placental microsomes yielded 19-hydroxy- and 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 [19-C3H2]- and [4-14C]19-hydroxyandrostenedione 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 C-19 hydroxylation by the placental aromatase complex. Radiometric kinetic analysis employing [19-C3H3]- and [1 beta,2 beta-3H]androstenedione as the comparative substrates provided evidence that the isotope effect is exerted solely through the Vmax 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.
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