Metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes. enzyme properties and kinetic parameters in the formation of estrogens and 17β-hydroxy-1,4-androstadien-3-one

Abstract

In this investigation the metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes was evaluated. The experiments were conducted with the substrate [ 3H]-1,4-androstadiene-3,17-dione, which was synthesized using a tritium-hydrogen keto-enol exchange technique. [4- 14C]-l,4-Androstadiene-3, 17-dione and [4- 14C]-17β-hydroxy-1,4-androstadien-3-one also were synthesized and used, together with [ 14C]-estrone and [ 14C]-estradiol-17β, as internal recovery standards for quantification. The radiolabeled metabolites identified from incubation mixtures with placental microsomes were estrone, estradiol-17β and 17β-hydroxy-1,4-androstadien-3-one; two other metabolites, one less polar than the substrate and the other more polar than estradiol-17β, were observed but not investigated further. The enzymatic conversions required either NADPH or NADH as cofactor and there was no apparent tritium loss in the transformation of substrate to products. The rates of formation of metabolites were linear with time for at least 30 min and with the amount of microsomal protein up to 15 mg. Tbe apparent pH optimum for placental microsomal estrogen synthetase was 7.5, and the apparent optimum pH for the 17β-hydroxysteroid oxidoreductase ranged from pH6.0 to pH7.4. The apparent optimal temperature for the estrogen synthetase activity was 37°C and for 17β-hydroxysteroid oxidoreductase it ranged from 37°C to 58°C. The apparent K m of estrogen synthetase for 1,4-androstadiene-3,17-dione was 1.3μM, and the apparent K m of 17β-hydroxysteroid oxidoreductase for 1,4-androstadiene-3, 17-dione was 24 μM. The apparent K m of estrogen synthetase for NADPH and NADH were 0.24 mM and 1.0mM, respectively; the apparent K m of placental microsomal 17β-hydroxysteroid oxidoreductase for NADH was 0.5 mM and that for NADPH was 0.67 mM. Androstenedione was found to be a competitive inhibitor of [ 3H]-1,4-androstadiene-3,17-dione aromatization ( K i ) = 0.75 μM androstenedione) and of 17β-hydroxysteroid oxidoreduction ( K i ) = 17μM androstenedione). Fifty per cent inhibition of estrogen synthetase and of 17β-hydroxysteroid oxidoreductase activities were obtained with an androstenedione concentration of 80 μM. The specific activity of NADPH-dependent cytochrome c reductase of placental microsomes was 22 nmol × mg −1 protein × min −1. The dependency of placental microsomal [ 3H]-1,4-androstadiene-3,17-dione aromatization on cytochrome P-450 and upon NADPH-dependent cytochrome c reductase was demonstrated using both anti-human and anti-porcine hepatic microsomal NADPH-dependent cytochrome c (P-450) reductase antibodies. The estrogen synthetase and NADPH-dependent cytochrome c reductase activities were inhibited greatly by the microsomal specific antibodies, whereas non-immune γ-globulin had little effect on enzyme activity. The 17β-hydroxysteroid oxidoreductase activity was affected only slightly by these antibodies.