Moore W R, Schatzman G L
Marion Merrell Dow Research Institute, Cincinnati, Ohio 45215.
J Biol Chem. 1992 Nov 5;267(31):22003-6.
A rapid and simple purification of milligram amounts of 2,3-oxidosqualene cyclase, an integral membrane enzyme that catalyzes the cyclization of squalene epoxide to lanosterol, is reported. Several nonionic detergents (Triton X-100, Tween 80, Emulphogene, and lauryl maltoside) were evaluated for solubilization of oxidosqualene cyclase from rat liver microsomes. At a detergent concentration of 5 mg/ml, lauryl maltoside was approximately 10 times more effective than Emulphogene in the solubilization of oxidosqualene cyclase; Triton X-100 and Tween 80 were less effective than Emulphogene as judged by the relative specific activities of the solubilized enzyme. Treatment of microsomes with lauryl maltoside resulted in a selective solubilization of the cyclase with concomitant activation of the enzyme. The solubilized enzyme was purified to homogeneity by fast protein liquid chromatography. The purified enzyme consists of a single subunit that has an apparent molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme obeys saturation kinetics and the apparent Km of (2,3)-oxidosqualene is 15 microM; the apparent kcat/Km is 200 M-1.min-1. An improved assay of the enzyme that utilizes high performance liquid chromatography methods is also described.
据报道，一种能催化角鲨烯环氧化物环化生成羊毛甾醇的整合膜酶——2,3-氧化角鲨烯环化酶，可实现毫克量的快速简便纯化。评估了几种非离子去污剂（Triton X-100、吐温80、乳化剂和月桂基麦芽糖苷）对大鼠肝脏微粒体中氧化角鲨烯环化酶的增溶作用。在去污剂浓度为5 mg/ml时，月桂基麦芽糖苷在氧化角鲨烯环化酶增溶方面的效果比乳化剂高约10倍；从增溶酶的相对比活性判断，Triton X-100和吐温80的效果不如乳化剂。用月桂基麦芽糖苷处理微粒体导致环化酶选择性增溶，同时酶被激活。通过快速蛋白质液相色谱法将增溶酶纯化至同质。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，纯化后的酶由一个亚基组成，表观分子量为65,000。该酶符合饱和动力学，(2,3)-氧化角鲨烯的表观Km为15 μM；表观kcat/Km为200 M-1·min-1。还描述了一种利用高效液相色谱法的改进酶测定方法。
