Purification and characterization of 3 alpha/beta-hydroxysteroid dehydrogenase from mature porcine testicular cytosol.
NADPH-dependent 3 alpha/beta-hydroxysteroid dehydrogenase (3 alpha/beta-HSD) was purified to apparent homogeneity from testicular cytosol of mature pigs. The purified enzyme catalyzes the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to both 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-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 3 alpha/beta-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 5 alpha- and 5 beta-DHT, 5 alpha- and 5 beta-dihydroprogesterone, 5 alpha- and 5 beta-dihydrocortisol, prostaglandin E2, 13,14-dihydro-15-keto-prostaglandin E2 and 13,14-dihydro-15-keto-prostaglandin F2 alpha. 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 AgNO3, SH-reagent, quercetin, hexesterol, stilbestrol, disulfiram and divalent cation such as Cu2+, Hg2+ and Cd2+. 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.
Nakajin
S
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo, Japan.
Fujita
Y
Y
Ohno
S
S
Uchida
M
M
Aoki
M
M
Shinoda
M
M
D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000818:Animals; D015153:Blotting, Western; D002413:Cations, Divalent; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D045166:Estradiol Congeners / Q000494:pharmacology; D007526:Isoelectric Point; D008297:Male; D008970:Molecular Weight; D010449:Peptide Mapping; D012835:Silver Nitrate / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine; D013737:Testis / Q000201:enzymology*; D020245:p-Chloromercuribenzoic Acid
D002413:Cations, Divalent; D002729:Chloromercuribenzoates; D004791:Enzyme Inhibitors; D045166:Estradiol Congeners; D020245:p-Chloromercuribenzoic Acid; D012835:Silver Nitrate; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)