Kinetic studies on androstenedione production in ovarian microsomes from immature rats

Androstenedione formation from progesterone by P-450(17 alpha,lyase) was investigated in ovarian microsomes of immature rats treated with pregnant mare serum gonadotropin. Successive monooxygenase reactions in the formation of androstenedione without the intermediate leaving P-450(17 alpha,lyase) were demonstrated by a double-substrate double-label experiment using [14C]progesterone and 17 alpha-[3H]hydroxyprogesterone as substrates and also by specific reduction in the concentration of intermediate 17 alpha-hydroxyprogesterone in the reaction medium by reaction of liposomal P-450C21. A detailed kinetic study on the reactions of P-450(17 alpha,lyase) in microsomes was conducted in the steady state. Kinetic parameters indicated the C17,C20-lyase reaction for 17 alpha-hydroxyprogesterone (Km = 80 nM) to be strongly inhibited by progesterone (Ki = 8 nM). In the presence of a high concentration of progesterone, as in the case of in vivo rat ovary, most androstenedione is concluded to be formed directly from progesterone by successive monooxygenase reactions catalyzed by P-450(17 alpha,lyase). 20 alpha-Dihydroprogesterone competitively inhibited the C17,C20-lyase reaction for 17 alpha-hydroxyprogesterone with Ki = 23 nM, but had only slight effect on progesterone metabolism to androstenedione. 20 alpha-Dihydroprogesterone, thus, cannot be a regulator for androstenedione formation in rat ovary.