3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.

A Stolz; H Takikawa; Y Sugiyama; J Kuhlenkamp; N Kaplowitz

doi:10.1172/JCI112829

3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.

A Stolz, H Takikawa, Y Sugiyama, J Kuhlenkamp, and N Kaplowitz

Published February 1, 1987 - More info

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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.

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