PURIFICATION AND CHARACTERIZATION OF HAMSTER LIVER MICROSOMAL 7-ALPHA-HYDROXYCHOLESTEROL DEHYDROGENASE - SIMILARITY TO TYPE-I 11-BETA-HYDROXYSTEROID DEHYDROGENASE

While studying the bile acid synthetic pathway of hamsters, we discove red an NADP(+)-dependent liver microsomal 7 alpha-hydroxycholesterol d ehydrogenase (7 alpha-HCD) activity that was not observed in rat liver microsomal fractions. The hamster liver microsomal 7 alpha-HCD was pu rified to homogeneity using 2',5'-ADP and cholic acid-agarose affinity chromatography. 7 alpha-HCD displayed a molecular weight of approxima tely 34,000 on SDS-polyacrylamide gel electrophoresis; it is an intrin sic membrane protein of the hamster liver endoplasmic reticulum and ex ists as a multimeric aggregate in pure form. Partial N-terminal amino acid sequence analysis showed that 7 alpha-HCD had high sequence simil arity to human Ilp-hydroxysteroid dehydrogenase (11 beta-HSD; 24/30 am ino acid identity). The K-m values for corticosterone and 7 alpha-hydr oxycholesterol were 1.2 and 1.9 mu M, respectively, for purified 7a-HC D; both reactions displayed identical V-max values (approximately 170 nmol/min/mg of protein). The IC50 of carbenoxolone, a competitive inhi bitor of 11 beta-HSD, was 75 nM for 7 alpha-hydroxycholesterol dehydro genation and 210 nM for corticosterone dehydrogenation. The tissue-spe cific expression in hamster was as follows: adrenal greater than or eq ual to liver > kidney > testis >> brain > lung. Microsomal 7 alpha-HCD is uniquely expressed in hamster liver and to some extent in human li ver but not in rat liver. Western blot analysis with two antibodies el icited against an N-terminal peptide of the human 11 beta-HSD and puri fied hamster liver 7a-HCD, respectively, suggested the presence of mul tiple forms of 7 alpha-HCD in hamster liver, most likely due to the ex istence of a family of 11 beta-HSD proteins. Since 7-oxocholesterol is a potent inhibitor of cholesterol 7 alpha-hydroxylase, alternative me chanisms for regulation of bile acid synthesis may exist in human and hamster liver due to production of this metabolite and its potential a s an oxysterol.