Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1987 Feb;79(2):427–434. 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, N Kaplowitz
PMCID: PMC424091  PMID: 3468121

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.

Full text

PDF
427

Images in this article

431Image
on p.431
431Image
on p.431

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aw T. Y., Ookhtens M., Ren C., Kaplowitz N. Kinetics of glutathione efflux from isolated rat hepatocytes. Am J Physiol. 1986 Feb;250(2 Pt 1):G236–G243. doi: 10.1152/ajpgi.1986.250.2.G236. [DOI] [PubMed] [Google Scholar]
  2. Batta A. K., Salen G., Shefer S. Transformation of bile acids into iso-bile acids by Clostridium perfringens: possible transport of 3 beta-hydrogen via the coenzyme. Hepatology. 1985 Nov-Dec;5(6):1126–1131. doi: 10.1002/hep.1840050611. [DOI] [PubMed] [Google Scholar]
  3. Berséus O. Conversion of cholesterol to bile acids in rat: purification and properties of a delta-4-3-ketosteroid 5-beta-reductase and a 3-alpha-hydroxysteroid dehydrogenase. Eur J Biochem. 1967 Nov;2(4):493–502. doi: 10.1111/j.1432-1033.1967.tb00163.x. [DOI] [PubMed] [Google Scholar]
  4. Björkhem I., Angelin B., Einarsson K., Ewerth S. Fasting levels of monoketonic bile acids in human peripheral and portal circulation. J Lipid Res. 1982 Sep;23(7):1020–1025. [PubMed] [Google Scholar]
  5. Björkhem I., Danielsson H., Wikvall K. Reduction of C19-, C21-, C24-, and C27-3-oxosteroids by rat-liver microsomes. Eur J Biochem. 1973 Jul 2;36(1):8–15. doi: 10.1111/j.1432-1033.1973.tb02878.x. [DOI] [PubMed] [Google Scholar]
  6. Blitzer B. L., Donovan C. B. A new method for the rapid isolation of basolateral plasma membrane vesicles from rat liver. Characterization, validation, and bile acid transport studies. J Biol Chem. 1984 Jul 25;259(14):9295–9301. [PubMed] [Google Scholar]
  7. ENEROTH P. THIN-LAYER CHROMATOGRAPHY OF BILE ACIDS. J Lipid Res. 1963 Jan;4:11–16. [PubMed] [Google Scholar]
  8. Glatt H. R., Cooper C. S., Grover P. L., Sims P., Bentley P., Merdes M., Waechter F., Vogel K., Guenthner T. M., Oesch F. Inactivation of a diol epoxide by dihydrodiol dehydrogenase but not by two epoxide hydrolases. Science. 1982 Mar 19;215(4539):1507–1509. doi: 10.1126/science.7038877. [DOI] [PubMed] [Google Scholar]
  9. Glatt H. R., Vogel K., Bentley P., Oesch F. Reduction of benzo(a)pyrene mutagenicity by dihydrodiol dehydrogenase. Nature. 1979 Jan 25;277(5694):319–320. doi: 10.1038/277319a0. [DOI] [PubMed] [Google Scholar]
  10. Ikawa S. 3-beta-hydroxy-C24-steroid dehydrogenase in rat liver microsomes. Yonago Acta Med. 1968 Dec;12(3):155–162. [PubMed] [Google Scholar]
  11. Ikeda M., Hattori H., Ikeda N., Hayakawa S., Ohmori S. Purification and characterization of the multiple forms of 3 alpha-hydroxysteroid dehydrogenase in rat liver cytosol. Hoppe Seylers Z Physiol Chem. 1984 Mar;365(3):377–391. doi: 10.1515/bchm2.1984.365.1.377. [DOI] [PubMed] [Google Scholar]
  12. Ikeda M., Hayakawa S., Ezaki M., Ohmori S. An NADP-dependent 3 alpha-hydroxysteroid dehydrogenase of rat liver active against C19, C20, C23, C24, C25 and C26 steroids. Hoppe Seylers Z Physiol Chem. 1981 May;362(5):511–520. doi: 10.1515/bchm2.1981.362.1.511. [DOI] [PubMed] [Google Scholar]
  13. Inoue M., Kinne R., Tran T., Arias I. M. Taurocholate transport by rat liver sinusoidal membrane vesicles: evidence of sodium cotransport. Hepatology. 1982 Sep-Oct;2(5):572–579. doi: 10.1002/hep.1840020510. [DOI] [PubMed] [Google Scholar]
  14. Jones D. P. Determination of pyridine dinucleotides in cell extracts by high-performance liquid chromatography. J Chromatogr. 1981 Oct 9;225(2):446–449. doi: 10.1016/s0378-4347(00)80293-5. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Meier P. J., St Meier-Abt A., Barrett C., Boyer J. L. Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles. Evidence for an electrogenic canalicular organic anion carrier. J Biol Chem. 1984 Aug 25;259(16):10614–10622. [PubMed] [Google Scholar]
  17. Moldéus P., Högberg J., Orrenius S. Isolation and use of liver cells. Methods Enzymol. 1978;52:60–71. doi: 10.1016/s0076-6879(78)52006-5. [DOI] [PubMed] [Google Scholar]
  18. Penning T. M., Mukharji I., Barrows S., Talalay P. Purification and properties of a 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs. Biochem J. 1984 Sep 15;222(3):601–611. doi: 10.1042/bj2220601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shefer S., Salen G., Hauser S., Dayal B., Batta A. K. Metabolism of iso-bile acids in the rat. J Biol Chem. 1982 Feb 10;257(3):1401–1406. [PubMed] [Google Scholar]
  20. Stolz A., Sugiyama Y., Kuhlenkamp J., Osadchey B., Yamada T., Belknap W., Balistreri W., Kaplowitz N. Cytosolic bile acid binding protein in rat liver: radioimmunoassay, molecular forms, developmental characteristics and organ distribution. Hepatology. 1986 May-Jun;6(3):433–439. doi: 10.1002/hep.1840060319. [DOI] [PubMed] [Google Scholar]
  21. Strange R. C. Hepatic bile flow. Physiol Rev. 1984 Oct;64(4):1055–1102. doi: 10.1152/physrev.1984.64.4.1055. [DOI] [PubMed] [Google Scholar]
  22. Sugiyama Y., Stolz A., Sugimoto M., Kaplowitz N. Evidence for a common high affinity binding site on glutathione S-transferase B for lithocholic acid and bilirubin. J Lipid Res. 1984 Nov;25(11):1177–1183. [PubMed] [Google Scholar]
  23. Sugiyama Y., Yamada T., Kaplowitz N. Newly identified bile acid binders in rat liver cytosol. Purification and comparison with glutathione S-transferases. J Biol Chem. 1983 Mar 25;258(6):3602–3607. [PubMed] [Google Scholar]
  24. TOMKINS G. M. A mammalian 3alpha-hydroxysteroid dehydrogenase. J Biol Chem. 1956 Jan;218(1):437–447. [PubMed] [Google Scholar]
  25. Takikawa H., Otsuka H., Beppu T., Seyama Y., Yamakawa T. Quantitative determination of bile acid glucuronides in serum by mass fragmentography. J Biochem. 1982 Oct;92(4):985–998. doi: 10.1093/oxfordjournals.jbchem.a134055. [DOI] [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Usui T., Kawamoto H., Sakamoto A., Katayama K. Studies on a 3alpha-hydroxysteroid dehydrogenase in rat liver. Yonago Acta Med. 1968 Aug;12(2):111–117. [PubMed] [Google Scholar]
  28. Vessey D. A., Zakim D. Inhibition of glutathione S-transferase by bile acids. Biochem J. 1981 Aug 1;197(2):321–325. doi: 10.1042/bj1970321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vogel K., Bentley P., Platt K. L., Oesch F. Rat liver cytoplasmic dihydrodiol dehydrogenase. Purification to apparent homogeneity and properties. J Biol Chem. 1980 Oct 25;255(20):9621–9625. [PubMed] [Google Scholar]
  30. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  31. Wörner W., Oesch F. Identity of dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase in rat but not in rabbit liver cytosol. FEBS Lett. 1984 May 21;170(2):263–267. doi: 10.1016/0014-5793(84)81325-3. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES