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 dehydroepiandro sterone (DHA) to 4-androstenedione and also for the conversion of 5,16 -androstadien-3 beta-ol (andien-beta) to 4,16-androstadien-3-one (dien one). Therefore, 3 beta-HSD-I plays an essential role in the biosynthe sis of hormonally and pheromonally active steroids. Previous studies f rom 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]. Th e aim of the present studies was to investigate the reactions further by examining the effects of two classical steroidal inhibitors of 3 be ta-HSD-I, trilostane (WIN 24540) and cyanoketone (WIN 19578), on the k inetic 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 k(m(app)) for DHA which at the highest concentration used, wer e 15-fold the control K-m(app) of 1.4 mu mol/l. No effect on the V-max (app) (6.55 +/- 0.74 nmol/h/mg protein) was observed, demonstrating th at competitive inhibition was evident. Slope and intercept replots con firmed the competitive nature of the inhibition and K-i(app) values of 0.16 mu mol/l for trilostane and 0.20 mu mol/l for cyanoketone were r espectively 9 and 7-fold lower than the K-m(app) value. In contrast, t rilostane and cyanoketone had no effect on the K-m(app) for andien-bet a (0.26 mu mol/l). The V-max(app) (1.12 nmol/h/mg protein) was decreas ed by 40-50% only by trilostane at the highest concentration used, dem onstrating a very low affinity for the andien-beta active site. K-i(ap p) values for trilostane and cyanoketone, obtained from slope and inte rcept replots were, respectively 1.1 and 1.6 mu mol/l, which were 4 an d B-fold greater than the K-m(app) for andien-beta. Therefore, trilost ane and cyanoketone were powerful competitive inhibitors of the conver sion of DHA to 4-androstenedione but were weak non-competitive inhibit ors of the conversion of andien-beta to dienone. The selective effects of trilostane and cyanoketone on the 3 beta-HSD-Is involved in the an drogen and 16-androstene biosynthetic pathways strongly suggest that t he reactions are catalysed by separate enzymes, or at least separate, non-interacting active sites on a single enzyme. Copyright (C) 1996 El sevier Science Ltd.