Purification and characterization of 3α/β-hydroxysteroid dehydrogenase from mature porcine testicular cytosol

Abstract

NADPH-dependent 3α/β-hydroxysteroid dehydrogenase (3α/β-HSD) was purified to apparent homogeneity from testicular cytosol of mature pigs. The purified enzyme catalyzes the conversion of 5α-dihydrotestosterone (5α-DHT) to both 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol. The molecular weight of the enzyme was estimated to be 31 kDa by SDS-polyacrylamide gel electrophoresis and 40 kDa by gel filtration chromatography indicating that the native 3α/β-HSD is a monomer. The isoelectric point of the purified enzyme was found to be 6.2 by density gradient isoelectric focusing and 6.4 by chromatofocusing. The enzyme reduced both 5α- and 5β-DHT, 5α- and 5β-dihydroprogesterone, 5α- and 5β-dihydrocortisol, prostaglandin E 2, 13,14-dihydro-15-keto-prostaglandin E 2 and 13,14-dihydro-15-keto-prostaglandin F 2α. Moreover, the enzyme caused rapid reduction of other carbonyl compounds including aldehydes, ketones and quinones. The rates of reduction of these compounds are fast relative to the rates of reduction of steroids and prostaglandins. The purified enzyme was inhibited by AgNO 3, SH-reagent, quercetin, hexesterol, stilbestrol, disulfiram and divalent cation such as Cu 2+, Hg 2+ and Cd 2+. The two enzymes show certain similarities (e.g. molecular weight, cross-reactivity to a common antibody) and certain striking differences (e.g. pI, effects of various inhibitors and greater enzyme activity towards steroids (neonatal form) or prostaglandins (mature form). Reasons are give for suggesting that these enzymes are closely related to carbonyl reductase.