3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.
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.
Stolz
A
A
Takikawa
H
H
Sugiyama
Y
Y
Kuhlenkamp
J
J
Kaplowitz
N
N
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
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