Purification and properties of 3 alpha-hydroxysteroid dehydrogenase as a 3-keto bile acid reductase from human liver cytosol.
The NADPH-dependent 3 alpha-hydroxysteroid dehydrogenases (peaks 1, 2 and 3) acting on 3-keto-5 beta-cholanoic acid separated by carboxymethyl-cellulose chromatography from human liver cytosol were purified to homogeneous protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using Affi-Gel blue, phenyl-Sepharose CL-4B, TSKgel G3000 SW chromatography and chromatofocusing. The overall purifications of the enzymes from cytosol were 316-fold (peak 1), 232-fold (peak 2) and 345-fold (peak 3) and the recoveries of the enzymes were 0.4% (peak 1), 7.1% (peak 2) and 3.7% (peak 3). The isoelectric points of the enzymes were found to be 7.34, 7.46 and 7.88 by chromatofocusing. The molecular weights of the enzymes were similar and estimated to be about 32,000 by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymological properties were nearly identical among the three forms. The reaction was reversible and the optimum pH of the enzymes for oxidation was about 8.4 and that for reduction was about 7.4. The enzymes could not reduce 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid, 7 alpha-hydroxy-5 beta-cholestan-3-one and 7 alpha, 12 alpha-dihydroxy-5 beta-cholestan-3-one to the corresponding 3 alpha-hydroxysteroids, whereas the enzymes could reduce 3,7-disubstituted 3-keto bile acids. Thus, the enzymes purified in this study were found to have a stereospecific character for some 3-ketosteroids. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of dithiothreitol to the reaction mixture, indicating that the enzyme required a sulfhydryl group for activity.
Third Department of Internal Medicine, Hyogo College of Medicine, Japan.
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K
Kudo
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Y
Amuro
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Hada
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Higashino
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001647:Bile Acids and Salts / Q000378:metabolism; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007651:Keto Acids / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity; D020245:p-Chloromercuribenzoic Acid
D000596:Amino Acids; D001647:Bile Acids and Salts; D002729:Chloromercuribenzoates; D007651:Keto Acids; D009249:NADP; D020245:p-Chloromercuribenzoic Acid; D015096:3-Hydroxysteroid Dehydrogenases