Purification and characterization of 5 alpha-dihydrotestosterone 3 beta-hydroxysteroid dehydrogenase from mature pig testicular cytosol.
NADPH-dependent 5 alpha-dihydrotestosterone 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was purified to apparent homogeneity from mature pig testicular cytosol. The purified enzyme catalyzed the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to 5 alpha-androstane-3 beta, 17 beta-diol. The molecular weight was estimated to be 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 28 kDa by gel filtration chromatography, indicating that the native 3 beta-HSD is a monomer. The isoelectric point of the purified enzyme was 5.8 as determined by chromatofocusing. The purified enzyme reduced not only 5 alpha-DHT but also 5 beta-DHT, 5 alpha(or 5 beta)-androstanedione, 5 alpha(or 5 beta)-dihydroprogesterone, prostaglandin E1, 13,14-dihydro-15-keto-prostaglandin F2 alpha, glyceladehyde, xylose and glucuronic acid. Moreover, the enzyme reduced other carbonyl compounds including aromatic aldehydes, aromatic ketones and quinones such as 4-nitrobenzaldehyde, 4-benzoylpyridine, phenylglyoxal, cyclohexanone and 9,10-phenanthrenequinone at high rates when compared with steroids, prostaglandins and sugars. The purified enzyme was inhibited by AgNO3, SH-reagent, disulfiram, hexesterol, stilbestrol, disulfiram and divalent cations such as Cu2+, Hg2+, Cd2+ and Co2+. Furthermore, the enzymatic properties of the purified enzyme, including catalytic activity, inhibitory effects by various agents and immunological properties, were compared with those of 3 alpha/beta-HSD enzymes from pig testicular cytosol.
Nakajin
S
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo, Japan.
Ishii
A
A
Shinoda
M
M
D015096:3-Hydroxysteroid Dehydrogenases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology*; D013196:Dihydrotestosterone / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008297:Male; D008970:Molecular Weight; D013379:Substrate Specificity; D013552:Swine; D013737:Testis / Q000201:enzymology*
D013196:Dihydrotestosterone; D015096:3-Hydroxysteroid Dehydrogenases