Identification of 7 alpha,12 alpha-dihydroxy-5 beta-cholestan-3-one 3 alpha-hydroxysteroid dehydrogenase.
A reductase catalyzing the reduction of the 3-ketone group of 7 alpha,12 alpha-dihydroxy-5 beta-cholestan-3-one and 7 alpha-hydroxy-5 beta-cholestan-3-one, which are the intermediates in the conversion of cholesterol to cholic acid and chenodeoxycholic acid, respectively, into the 3 alpha-hydroxyl group, was purified about 250-fold as judged by the activity from the 100,000 X g supernatant of rat liver homogenate. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretography was 32,000. The absorption spectrum of the purified enzyme showed only a peak at 280 nm due to aromatic amino acids, precluding the presence of a chromophoric prosthetic group in the molecule. The enzyme showed activity toward a variety of substrates, including 3-oxo-5 beta-cholanoic acid, androsterone, 9,10-phenanthrenquinone, p-nitrobenzaldehyde, but not toward glucuronic acid, DL-glyceraldehyde, and glycolaldehyde. The optimal pH for the reduction of 7 alpha-hydroxy-5 beta-cholestan-3-one was 7.4, and the cofactor required was either NADPH or NADH, though the former gave the higher activity. Judging from the chromatography behavior as well as substrate specificity, the enzyme was identified as 3 alpha-hydroxysteroid dehydrogenase (3 alpha-hydroxysteroid:NAD(P)+ oxidoreductase, EC 1.1.1.50).
Usui
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Okuda
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D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010088:Oxidoreductases / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D009243:NAD; D009249:NADP; D010088:Oxidoreductases; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); C021936:3-oxo-5 beta-steroid delta 4-dehydrogenase