Differential effects of trilostane and cyanoketone on the 3 beta-hydroxysteroid dehydrogenase-isomerase reactions in androgen and 16-androstene biosynthetic pathways in the pig testis.
3 beta-Hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) activity in the pig testis is responsible for the conversion of dehydroepiandrosterone (DHA) to 4-androstenedione and also for the conversion of 5,16-androstadien-3 beta-ol (andien-beta) to 4, 16-androstadien-3-one (dienone). Therefore, 3 beta-HSD-I plays an essential role in the biosynthesis of hormonally and pheromonally active steroids. Previous studies from this laboratory have suggested that the 3 beta-HSD-I reactions in the androgen and 16-androstene biosynthetic pathways may be catalysed by different enzymes with selective substrate specificities [3, 4]. The aim of the present studies was to investigate the reactions further by examining the effects of two classical steroidal inhibitors of 3 beta-HSD-I, trilostane (WIN 24540) and cyanoketone (WIN 19578), on the kinetic parameters of the 3 beta-HSD-I reactions in immature (< 3 weeks) pig testis microsomes. In kinetic analyses of the conversion of DHA to 4-androstenedione, both trilostane and cyanoketone caused increases in the Km(app) for DHA which at the highest concentration used, were 15-fold the control Km(app) of 1.4 mumol/l. No effect on the Vmax(app) (6.55 +/- 0.74 nmol/h/mg protein) was observed, demonstrating that competitive inhibition was evident. Slope and intercept replots confirmed the competitive nature of the inhibition and Ki(app) values of 0.16 mumol/l for trilostane and 0.20 mumol/l for cyanoketone were respectively 9 and 7-fold lower than the Km(app) value. In contrast, trilostane and cyanoketone had no effect on the Km(app) for andien-beta (0.26 mumol/l). The Vmax(app) (1.12 nmol/h/mg protein) was decreased by 40-50% only by trilostane at the highest concentration used, demonstrating a very low affinity for the andien-beta active site. Ki(app) values for trilostane and cyanoketone, obtained from slope and intercept replots were, respectively 1.1 and 1.6 mumol/l, which were 4 and 6-fold greater than the Km(app) for andien-beta. Therefore, trilostane and cyanoketone were powerful competitive inhibitors of the conversion of DHA to 4-androstenedione but were weak non-competitive inhibitors of the conversion of andien-beta to dienone. The selective effects of trilostane and cyanoketone on the 3 beta-HSD-Is involved in the androgen and 16-androstene biosynthetic pathways strongly suggest that the reactions are catalysed by separate enzymes, or at least separate, non-interacting active sites on a single enzyme.
Cooke
G M
GM
Toxicology Research Division, Health Canada, Ottawa, Ontario, Canada.
D000730:Androstadienes / Q000378:metabolism; D000735:Androstenedione / Q000096:biosynthesis; D000736:Androstenes / Q000378:metabolism*; D000737:Androstenols / Q000378:metabolism; D000818:Animals; D003489:Cyanoketone / Q000494:pharmacology*; D003687:Dehydroepiandrosterone / Q000378:metabolism; D013196:Dihydrotestosterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D011375:Progesterone Reductase / Q000037:antagonists & inhibitors*; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors*; D013552:Swine; D013737:Testis / Q000201:enzymology / Q000378:metabolism*
C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D000730:Androstadienes; D000736:Androstenes; D000737:Androstenols; D009097:Multienzyme Complexes; D013196:Dihydrotestosterone; C000152:androsta-5,16-dien-3 beta-ol; C042468:16-androstene; C003524:4,16-androstadien-3-one; D000735:Androstenedione; D003489:Cyanoketone; D003687:Dehydroepiandrosterone; D011375:Progesterone Reductase; D013251:Steroid Isomerases; C009954:trilostane