Song W, Chen J, Dean W L, Redinger R N, Prough R A
Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky 40292, USA.
J Biol Chem. 1998 Jun 26;273(26):16223-8. doi: 10.1074/jbc.273.26.16223.
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.
在研究仓鼠胆汁酸合成途径时，我们发现了一种NADP⁺依赖性肝微粒体7α-羟基胆固醇脱氢酶（7α-HCD）活性，而在大鼠肝微粒体组分中未观察到这种活性。使用2',5'-ADP和胆酸-琼脂糖亲和色谱法将仓鼠肝微粒体7α-HCD纯化至同质。在SDS-聚丙烯酰胺凝胶电泳上，7α-HCD的分子量约为34,000；它是仓鼠肝内质网的一种内在膜蛋白，以多聚体聚集体的形式纯在。部分N端氨基酸序列分析表明，7α-HCD与人类11β-羟基类固醇脱氢酶（11β-HSD；24/30氨基酸同一性）具有高度序列相似性。纯化的7α-HCD对皮质酮和7α-羟基胆固醇的Km值分别为1.2和1.9μM；两种反应显示出相同的Vmax值（约170 nmol/分钟/毫克蛋白质）。11β-HSD的竞争性抑制剂甘草次酸对7α-羟基胆固醇脱氢的IC50为75 nM，对皮质酮脱氢的IC50为210 nM。仓鼠中的组织特异性表达如下：肾上腺≥肝脏＞肾脏＞睾丸＞＞大脑＞肺。微粒体7α-HCD在仓鼠肝脏中独特表达，在人类肝脏中也有一定程度的表达，但在大鼠肝脏中不表达。分别用针对人类11β-HSD的N端肽和纯化的仓鼠肝脏7α-HCD产生的两种抗体进行蛋白质印迹分析表明，仓鼠肝脏中存在多种形式的7α-HCD，最可能是由于存在11β-HSD蛋白家族。由于7-氧代胆固醇是胆固醇7α-羟化酶的有效抑制剂，由于这种代谢产物的产生及其作为氧化甾醇的潜力，人类和仓鼠肝脏中可能存在胆汁酸合成调节的替代机制。
