Purification of 3 alpha-hydroxysteroid and 3 beta-hydroxysteroid dehydrogenases from human liver cytosol.
We previously reported that the human Y' bile acid binder, which has higher bile acid binding affinities than rat Y' binders (3 alpha-hydroxysteroid dehydrogenases), has dihydrodiol dehydrogenase activity and is different from 3 alpha-hydroxysteroid dehydrogenases. In this study, 3 alpha-hydroxysteroid dehydrogenases and 3 beta-hydroxysteroid dehydrogenase were purified from human liver, and bile acid binding affinities and enzyme kinetics of the 3 alpha-hydroxysteroid dehydrogenases were studied. On chromatofocusing of pooled Affigel blue fraction of the Y' fraction, three 3 alpha-hydroxysteroid dehydrogenase peaks eluted at pH 6.0, 5.7 and 5.4. These peaks did not bind bile acids, and further purification by hydroxyapatite-high-performance liquid chromatography gave pure 3 alpha-hydroxysteroid dehydrogenases with identical M(r) (36,000) having dihydrodiol dehydrogenase activity. 3 beta-Hydroxysteroid dehydrogenase was eluted together with Y' bile acid binder at pH 7.2 on chromatofocusing and was separated from Y' bile acid binder on hydroxyapatite-high-performance liquid chromatography as a pure protein with M(r) 32,000. The apparent Kms of 3 alpha-hydroxysteroid dehydrogenases were similar to those of rat enzymes. In conclusion, we purified human hepatic 3 alpha-hydroxysteroid dehydrogenases, which have similar characteristics to rat enzymes, but do not bind bile acids or reduce bile acid precursors. These data further support the importance of human bile acid binder in intracellular bile acid transport in the human liver.
Takikawa
H
H
Department of Medicine, Teikyo University School of Medicine, Tokyo, Japan.
Fujiyoshi
M
M
Nishikawa
K
K
Yamanaka
M
M
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D001647:Bile Acids and Salts / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*
D001647:Bile Acids and Salts; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)