Purification and characterization of hamster liver microsomal 7alpha-hydroxycholesterol dehydrogenase. Similarity to type I 11beta-hydroxysteroid dehydrogenase

J Biol Chem. 1998 Jun 26;273(26):16223-8. doi: 10.1074/jbc.273.26.16223.

Abstract

While studying the bile acid synthetic pathway of hamsters, we discovered an NADP+-dependent liver microsomal 7alpha-hydroxycholesterol dehydrogenase (7alpha-HCD) activity that was not observed in rat liver microsomal fractions. The hamster liver microsomal 7alpha-HCD was purified to homogeneity using 2', 5'-ADP and cholic acid-agarose affinity chromatography. 7alpha-HCD displayed a molecular weight of approximately 34,000 on SDS-polyacrylamide gel electrophoresis; it is an intrinsic membrane protein of the hamster liver endoplasmic reticulum and exists as a multimeric aggregate in pure form. Partial N-terminal amino acid sequence analysis showed that 7alpha-HCD had high sequence similarity to human 11beta-hydroxysteroid dehydrogenase (11beta-HSD; 24/30 amino acid identity). The Km values for corticosterone and 7alpha-hydroxycholesterol were 1.2 and 1.9 microM, respectively, for purified 7alpha-HCD; both reactions displayed identical Vmax values (approximately 170 nmol/min/mg of protein). The IC50 of carbenoxolone, a competitive inhibitor of 11beta-HSD, was 75 nM for 7alpha-hydroxycholesterol dehydrogenation and 210 nM for corticosterone dehydrogenation. The tissue-specific expression in hamster was as follows: adrenal >/= liver > kidney > testis >> brain > lung. Microsomal 7alpha-HCD is uniquely expressed in hamster liver and to some extent in human liver but not in rat liver. Western blot analysis with two antibodies elicited against an N-terminal peptide of the human 11beta-HSD and purified hamster liver 7alpha-HCD, respectively, suggested the presence of multiple forms of 7alpha-HCD in hamster liver, most likely due to the existence of a family of 11beta-HSD proteins. Since 7-oxocholesterol is a potent inhibitor of cholesterol 7alpha-hydroxylase, alternative mechanisms for regulation of bile acid synthesis may exist in human and hamster liver due to production of this metabolite and its potential as an oxysterol.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 11-beta-Hydroxysteroid Dehydrogenases
  • Amino Acid Sequence
  • Animals
  • Carbenoxolone / pharmacology
  • Catalysis
  • Cricetinae
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Hydroxysteroid Dehydrogenases / chemistry*
  • Hydroxysteroid Dehydrogenases / isolation & purification*
  • In Vitro Techniques
  • Kinetics
  • Male
  • Mesocricetus
  • Microsomes, Liver / enzymology*
  • Molecular Sequence Data
  • Rabbits
  • Rats
  • Rats, Sprague-Dawley

Substances

  • Enzyme Inhibitors
  • Hydroxysteroid Dehydrogenases
  • 11-beta-Hydroxysteroid Dehydrogenases
  • 7 alpha-hydroxysteroid dehydrogenase
  • Carbenoxolone