Purification and characterization of hamster liver microsomal 7alpha-hydroxycholesterol dehydrogenase. Similarity to type I 11beta-hydroxysteroid dehydrogenase.
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.
Song
W
W
Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky 40292, USA.
Chen
J
J
Dean
W L
WL
Redinger
R N
RN
Prough
R A
RA
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000595:Amino Acid Sequence; D000818:Animals; D002229:Carbenoxolone / Q000494:pharmacology; D002384:Catalysis; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification*; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley
D004791:Enzyme Inhibitors; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; C025376:7 alpha-hydroxysteroid dehydrogenase; D002229:Carbenoxolone