3 alpha-hydroxysteroid dehydrogenase activity catalyzed by purified pig adrenal 20 alpha-hydroxysteroid dehydrogenase.
In earlier studies, two distinct molecules, 20 alpha-HSD-I and 20 alpha-HSD-II, responsible for 20 alpha-HSD activity of pig adrenal cytosol were purified to homogeneity and characterized [S. Nakajin et al., J. Steroid Biochem. 33 (1989) 1181-1189]. We report here that the purified 20 alpha-HSD-I, which mainly catalyzes the reduction of 17 alpha-hydroxyprogesterone to 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, catalyzes 3 alpha-hydroxysteroid oxidoreductase activity for 5 alpha (or 5 beta)-androstanes (C19), 5 alpha (or 5 beta)-pregnanes (C21) in the presence of NADPH as the preferred cofactor. The purified enzyme has a preference for the 5 alpha (or 5 beta)-androstane substrates rather than 5 alpha (or 5 beta)-pregnane substrates, and the 5 beta-isomers rather than 5 alpha-isomers, respectively. Kinetic constants in the reduction for 5 alpha-androstanedione (Km; 3.3 microM, Vmax; 69.7 nmol/min/mg) and 5 beta-androstanedione (Km; 7.7 microM, Vmax; 135.7 nmol/min/mg) were demonstrated for comparison with those for 17 alpha-hydroxyprogesterone (Km; 26.2 microM, Vmax; 1.3 nmol/min/mg) which is a substrate for 20 alpha-HSD activity. Regarding oxidation, the apparent Km and Vmax values for 3 alpha-hydroxy-5 alpha-androstan-17-one were 1.7 microM and 43.2 nmol/min/mg, and 1.2 microM and 32.1 nmol/min/mg for 3 alpha-hydroxy-5 beta-androstan-17-one, respectively. 20 alpha-HSD activity in the reduction of 17 alpha-hydroxyprogesterone catalyzed by the purified enzyme was inhibited competitively by addition of 5 alpha-DHT with a Ki value of 2.0 microM. Furthermore, 17 alpha-hydroxyprogesterone inhibited competitively 3 alpha-HSD activity with a Ki value of 150 microM.
Nakajin
S
S
Department of Biochemistry, Faculty of Pharmaceutical Science, Hoshi University, Tokyo, Japan.
Fujii
S
S
Ohno
S
S
Shinoda
M
M
D015089:20-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D013196:Dihydrotestosterone / Q000378:metabolism; D007700:Kinetics; D013379:Substrate Specificity; D013552:Swine
D013196:Dihydrotestosterone; D015096:3-Hydroxysteroid Dehydrogenases; D015089:20-Hydroxysteroid Dehydrogenases