Cofactor Dependency of Soluble 3α-Hydroxysteroid Oxidoreductases in Rat Testis, Prostate, and Epididymis*

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Abstract
The present paper demonstrates the presence of at least two 3-hydroxysteroid oxidoreductases (3-HSO) in rat testis, prostate, and epididymis cytosol preparations. The enzymes were either NADH or NADPH dependent. Further investigation by Sephadex G-200 chromatography revealed the presence of enzyme activities in the void volume (peak 1) and also eluting close to the methyl-carbonic anhydrase standard (mol wt, 34,000; peak 2). Enzyme activity in peak 1 was predominantly stimulated by NADH and that in peak 2 was stimulated mainly by NADPH. Both 3α- and 3β-HSO activities were observed in testicular eluates from peak 1; 3β-Adiol accounted for up to 50% of the 5aandrostanediols formed. In peak 2, 3α-HSO constituted more than 90% of the enzyme activity. In contrast, the prostatic and epididymal eluates revealed only 3α-HS0 activity; 3β-Adiol constituted less than 5% of the 5α-androstanediols formed in either peak 1 or 2. The apparent Km values for enzyme activation reveal differences in sensitivity to cofactors for enzymes in peaks 1 and 2 and also among testis, epididymis, and prostate. NADH caused a very similar activation of enzyme activity in peak 1 of the prostate and epididymis (Km, 50–100 μM), whereas the enzyme in the testis was activated by much lower cofactor concentration (Km, 5 μM). It is possible that this enzyme activity may represent microsomal contamination. The enzyme activity in peak 2 revealed very similar sensitivity to NADPH in all three organs (Km, 0.6–1.7 μM), confirming previous studies from our laboratory that the soluble NADPH-dependent enzymes in all three tissues are very similar, if not identical.