Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.1.1.3 (HSD)
 3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Purification and characterization of hamster liver microsomal 7alpha-hydroxycholesterol dehydrogenase. Similarity to type I 11beta-hydroxysteroid dehydrogenase. 963 80

11beta-Hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders, including insulin resistance and obesity. Because 11beta-HSD 1 is a membrane protein with a very hydrophobic character, it is difficult to purify it in an active state. Not much is known about the topological and structural determinants of 11beta-HSD 1, although the elucidation of the structure of 11beta-HSD 1 would be a great advantage in identifying specific 11beta-HSD 1 inhibitors. Bacterial expression of full-length or truncated 11beta-HSD 1 forms only led to insoluble proteins or to low amounts of enzyme, not sufficient for crystallization. Recently, we reported that the solubility of 11beta-HSD 1 could be increased by substitution of hydrophobic amino acid residues with arginine without affecting activity. Unfortunately, these truncated and soluble forms of 11beta-HSD 1 exhibited an unstable activity that declined very rapidly. So far, the proteins obtained were not suitable for crystallization. To obtain 11beta-HSD 1 in an active and soluble state, in the present investigation we focused on the amino acid sequence encoded by the first exon. Using bacterial and yeast expression systems, we found that this N-terminal peptide could be divided into two parts that have functions other than to anchor 11beta-HSD 1 into the ER membrane. The first hydrophobic part, consisting of amino acid residues 1-15, represents the membrane spanning domain and anchors 11beta-HSD 1 in the ER membrane. The second hydrophilic part of the peptide, consisting of amino acid residues 16-30, plays a crucial role in stabilizing the catalytic domain of 11beta-HSD 1 and in addition, acts as a spacer to keep the catalytic domain of 11beta-HSD 1 into the lumen of the ER. Evidently, we found that the hydrophilic amino acids 24-30 determine 11beta-HSD 1 enzyme activity. Combined, all information obtained should help to design an optimal 11beta-HSD 1 enzyme in the near future with all desired attributes: soluble, active and easy to obtain and purify in sufficient amounts. This soluble and active 11beta-HSD 1 form should be the basis for our ongoing project, which is the determination of the three dimensional structure of 11beta-HSD 1.
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PMID:The critical role of the N-terminus of 11beta-hydroxysteroid dehydrogenase type 1, as being encoded by exon 1, for enzyme stabilization and activity. 1260 33