title: Partial purification and characterization of 7 alpha-hydroxysteroid dehydrogenase from rat liver microsomes.
abstract: An NADPH-dependent 7 alpha-hydroxysteroid dehydrogenase acting on 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was partially purified 160-fold with a yield of 13% from rat liver microsomes using DEAE-cellulose, hydroxyapatite and Affi-Gel Blue column chromatography. The specific activity of the purified enzyme was 91.3 nmol chenodeoxycholic acid formed/min per mg of protein. The reaction was reversible, and the optimum pH of the enzyme for the oxidation was about 8.5, whereas that for the reduction was about 5.0 A molecular weight of the enzyme was estimated to be about 130,000 by Superose 6TM gel filtration chromatography. The apparent Km value for 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was 35.7 microM and that for NADPH was 90.9 microM. The preferred substrate for the enzyme was 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid rather than 3 alpha,12 alpha-dihydroxy-7-keto-5 beta-cholanoic acid, a 7-keto-bile acid analogue. The enzyme also preferred the unconjugated form to the conjugated forms. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of reduced form of glutathione to the reaction mixture, indicating that the enzyme requires a sulfhydryl group for activity.
authors.forename: Y
authors.initials: Y
authors.lastname: Amuro
authors.forename: W
authors.initials: W
authors.lastname: Yamade
authors.forename: T
authors.initials: T
authors.lastname: Yamamoto
authors.forename: A
authors.initials: A
authors.lastname: Maebo
authors.forename: T
authors.initials: T
authors.lastname: Hada
authors.forename: K
authors.initials: K
authors.lastname: Higashino
mesh_terms: D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D020245:p-Chloromercuribenzoic Acid
chemical_list: D002729:Chloromercuribenzoates; D020245:p-Chloromercuribenzoic Acid; D006913:Hydroxysteroid Dehydrogenases; C025376:7 alpha-hydroxysteroid dehydrogenase