Studies on the Human Testis. VI. NADH-Linked Reactions of Microsomal Steroid 20α- and 20β-Hydroxysteroid Dehydrogenase and 17α-Hydroxylase*

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Abstract
NADH-linked 20α- and 20β-hydroxysteroid dehydrogenase and 17α-hydroxylase activities were demonstrated in the microsomal fraction of the human testis. The microsomal 20α-hydroxysteroid dehydrogenase showed substrate affinity to pregnenolone and progesterone and not to 17α-hydroxyprogesterone and preferred NADH to NADPH as a hydrogen donor. In the presence of NADH, the optimal pH for the enzyme was 7.7 and the apparent Michaelis constants of the enzyme for progesterone and pregnenolone at 37 C and pH 7.4 were 6.9–7.1 × 10−6M and in the order of 10−5M, respectively. 17α, 20β-Dihydroxypregn-4-en-3-one was the only significant metabolite produced from 17α-hydroxyprogesterone by microsomal fraction of the human testis in the presence of NADH. The apparent Michaelis constant of microsomal 20β-hydroxysteroid dehydrogenase for 17α-hydroyprogesterone in the presence of NADH was in the order of 10−5M at 37 C and pH 7.4. The microsomal 17α-hydroxylase catalyzed the metabolism of pregnenolone and progesterone at a similar rate in the presence of NADH. The optimal pH and the apparent Michaelis constant at 37 C and pH 7.4 of the NADH-linked reaction of 17α-hydroxylase for progesterone were 7.7 and 5.3–5.4 × 10−7M, respectively. The NADH-linked enzyme activity for progesterone was competitively inhibited by both pregn-5-ene-3β, 20α-diol (inhibition constant: 1.7 × 10−7M) and 20α-hydroxypregn-4-en-3-one (inhibition constant: 6.6 × 10−7M), and was resistant to poor oxygen supply during incubation. The results indicate that the microsomal 20α-hydroxysteroid dehydrogenase is a different enzyme from the one in the soluble fraction of the human testis and that microsomal 17α-hydroxylase in the human testis is activated by NADH as well as NADPH.

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