影响CYP27A1酶活性的机体内蛋白酶初探.docVIP

 n 影响 CYP27A1酶活性的机体内蛋白酶初探# 李晓波，殷莲华** （复旦大学上海医学院生理与病理生理学系，上海 200032） 5 10 15 20 摘要：目的：线粒体胆固醇 27-羟化酶（CYP27A1）是一种多功能细胞色素 P450 羟化酶，催 化胆汁酸生物合成酸性途径的第一步，即胆固醇 27 位羟基化反应，在维持细胞内胆固醇平 衡中发挥重要作用。本研究对机体内影响 CYP27A1 酶活性的蛋白酶进行初步探讨。方法：分 离和培养原代大鼠肝细胞，从大鼠肝脏组织及肝细胞中分离富含线粒体组分、细胞浆和粗制 富含溶酶体组分，在试管中进行胆固醇 27-羟化酶反应，并用 HPLC 分析胆固醇 27-羟化酶反 应产物。结果：分离的肝脏细胞浆可以使 CYP27A1 酶活性增加 2-4 倍, 煮沸灭活抑制该效应。 分离的溶酶体释放出的蛋白酶可以刺激 27-羟胆固醇的合成。而 EDTA 和蛋白酶抑制剂 AEBSF 可以降低 27-羟胆固醇生成，而二价钙离子则可以刺激其生成。结论：某种钙离子依赖的金 属蛋白酶和某种丝氨酸蛋白酶都有可能是刺激 CYP27A1 活性的蛋白酶, 蛋白酶水解反应可能 与控制线粒体胆固醇跨膜转运以及胆汁酸酸性合成途径有关。 关键词：病理学；CYP27A1；线粒体；胆固醇 中图分类号： R363.2 Proteolysis Stimulates Activity of Sterol 27-Hydroxylase (CYP27A1) in Mitochondria Li Xiaobo, Yin Lianhua (Department of Physiology and Pathophysiology, Fudan University Shanghai Medical College, Shanghai 200032) Abstract: Mitochondrial cholesterol 27-hydroxylase (CYP27A1; EC1.14.13.15) is a multifunctional P450 enzyme that catalyzes the initial reaction, 27-hydroxylation of cholesterol in 25 30 35 40 the “acidic” pathway of bile acid biosynthesis. CYP27A1 also plays several important roles in cholesterol homeostasis and affects atherogenesis. We already observed proteinase k stimulated CYP27A1 specific activity dramatically. Proteinases are located in the cells comprehensively, which is around mitochondria too. Incubation of isolated mitochondria with the cytosolic fraction stimulated CYP27A1 specific activity 2-4 fold. Heat-inactivation of the cytosolic fraction prevented this increase. Isolated crude lysosomal fraction stimulated the production of 27-hydroxycholesterol. However, EDTA and preteinase inhibitor AEBSF suppressed CYP27A1 activity, while CaCl2 perform the opposite effect. These findings suggest that protelysis is involved in the control of transmembrane mitochndrial cholesterol movement and, therefore, in the “acidic” pathway of bile acid biosynthesis. Key words: Pathophysiology; CYP27A1; mitochondria; cholesterol 0 引言 胆汁酸合成有两个途径，主要途径也称中性合成途径，首先由该通路的关键酶，胆固醇 7?-羟化酶（CYP7A1），催化胆固醇合成 7??羟化胆固