title: 3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.
abstract: Rat Y' bile acid binders (33 kD) have been previously recognized as cytosolic bile acid binding proteins (Sugiyama, Y., T. Yamada, and N. Kaplowitz, 1983, J. Biol. Chem., 258:3602-3607). We have now determined that these Y' binders are 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSD), bile acid-metabolizing enzymes. 3 alpha-HSD activity copurified with lithocholic acid-binding activity after sequential gel filtration, chromatofocusing, and affinity chromatography. Three peaks of 3 alpha-HSD activity (I, II, III) were observed in chromatofocusing and all were identified on Western blot by a specific Y' binder antiserum. 3 alpha-HSD-I, the predominant form, was purified and functioned best as a reductase at pH 7.0 with a marked preference for NADPH. Michaelis constant values for mono- and dihydroxy bile acids were 1-2 microM, and cholic acid competitively inhibited the reduction of 3-oxo-cholic acid. Under normal redox conditions, partially purified 3 alpha-HSD-I and freshly isolated hepatocytes catalyzed the rapid reduction of 3-oxo-cholic to cholic acid without formation of isocholic acid, whereas the reverse reaction was negligible. The Y' bile acid binders are therefore 3 alpha-HSD, which preferentially and stereospecifically catalyze the reduction of 3-oxo-bile acids to 3 alpha-hydroxy bile acids.
authors.forename: A
authors.initials: A
authors.lastname: Stolz
authors.forename: H
authors.initials: H
authors.lastname: Takikawa
authors.forename: Y
authors.initials: Y
authors.lastname: Sugiyama
authors.forename: J
authors.initials: J
authors.lastname: Kuhlenkamp
authors.forename: N
authors.initials: N
authors.lastname: Kaplowitz
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D051381:Rats; D013379:Substrate Specificity
chemical_list: D002352:Carrier Proteins; D008562:Membrane Glycoproteins; C037643:bile acid binding proteins; D015096:3-Hydroxysteroid Dehydrogenases; D006913:Hydroxysteroid Dehydrogenases; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D005982:Glutathione Transferase