3α-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. We have now determined that these Y' binders are 3α-hydroxysteroid dehydrogenases (3α-HSD), bile acid-metabolizing enzymes. 3α-HSD activity copurified with lithocholic acid-binding activity after sequential gel filtration, chromatofocusing, and affinity chromatography. Three peaks of 3α-HSD activity (I, II, III) were observed in chromatofocusing and all were identified on Western blot by a specific Y' binder antiserum. 3α-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 μM, and cholic acid competitively inhibited the reduction of 3-oxo-cholic acid. Under normal redox conditions, partially purified 3α-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 3α-HSD, which preferentially and stereospecifically catalyze the reduction of 3-oxo-bile acids to 3α-hydroxy bile acids.