Partial purification and characterization of 7α-hydroxysteroid dehydrogenase from rat liver microsomes

Abstract An NADPH-dependent 7α-hydroxysteroid dehydrogenase acting on 3α-hydroxy-7-keto-5β-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 130000 by Superose 6 TM gel filtration chromatography. The apparent K m value for 3α-hydroxy-7-keto-5β-cholanoic acid was 35.7 μM and that for NADPH was 90.9 μM. The preferred substrate for the enzyme was 3α-hydroxy-7-keto-5β-cholanoic acid rather than 3α,12α-dihydroxy-7-keto-5β-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 Pifp-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.