Enzyme activity assay for cholesterol 27-hydroxylase in mitochondria
Enzyme activity assay for cholesterol 27-hydroxylase in mitochondria
复制标题
线粒体胆固醇27-羟化酶的酶活性测定
DOI：
10.1194/jlr.m600117-jlr200
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发表时间：
2006-07-01
影响因子：
6.5
通讯作者：
Ren, Shunlin
中科院分区：
文献类型：
--
作者：
Li, Xiaobo;Hylemon, Philip;Ren, Shunlin
线粒体胆固醇27-羟化酶（CYP27A1）在维持细胞内胆固醇稳态中起重要作用。胆固醇传递到线粒体内膜被认为是胆汁酸合成“酸性”途径的限速步骤。这项工作报道了线粒体的蛋白酶K处理显著增加CYP27A1特异性活性。对于内源性线粒体胆固醇，用蛋白酶K处理可使CYP27A1特异性活性增加5倍。此外，在b-环糊精加蛋白酶K处理中添加外源胆固醇使其比活性提高了7倍。动力学研究表明，活性的增加与时间、蛋白酶K和底物浓度有关。蛋白酶K处理使CYP27A1对胆固醇的表观Km从400 mM降低到150 mM。通过这项新的实验，我们发现在大鼠肝细胞制备和细胞培养过程中，与从大鼠肝组织中新鲜分离的线粒体相比，线粒体逐渐失去CYP27A1活性。
Mitochondrial cholesterol 27-hydroxylase (CYP27A1) plays an important role in the maintenance of intracellular cholesterol homeostasis. Cholesterol delivery to the mitochondrial inner membrane is believed to be a rate-limiting step for the "acidic" pathway of bile acid synthesis. This work reports that proteinase K treatment of mitochondria markedly increases CYP27A1 specific activity. With endogenous mitochondrial cholesterol, treatment with proteinase K increased CYP27A1 specific activity by 5-fold. Moreover, the addition of the exogenous cholesterol in b-cyclodextrin plus proteinase K treatment increased the specific activity by 7-fold. Kinetic studies showed that the increased activity was time-, proteinase K-, and substrate concentration-dependent. Proteinase K treatment decreased the apparent Km of CYP27A1 for cholesterol from 400 to 150 mM. Using this new assay, we found that during rat hepatocyte preparation and cell culture, mitochondria gradually lose CYP27A1 activity compared with mitochondria freshly isolated from rat liver tissue.