MEMBRANE BINDING OF CHOLESTEROL OXIDASE

本研究的目的是研究胆固醇氧化酶与细胞膜的结合。胆固醇氧化酶是由一些革兰氏阴性细菌产生的，这些细菌可以将胆固醇作为碳源。其中一些微生物是致病性的，如马红球菌和结核分枝杆菌。有人提出，两个表面残基M332和W333通过将其侧链插入膜的疏水核心来促进结合。为了验证这一假设，将制造以下突变酶；M332A、M332C、W333A、W333C、W333F。Cys突变体将用荧光报告基团丙烯醛和单溴锰进行标记。将检测突变酶在不同离子强度和ph条件下与不同脂质组成的囊泡结合的能力。还将测试突变酶在胶束和囊泡中对胆固醇的活性。从这些研究中获得的信息将有助于改善血清胆固醇测定，了解膜胆固醇在受体功能中的作用，并揭示胆固醇氧化酶作为潜在毒力因子的作用。

The aim of this research is to investigate the binding of cholesterol oxidase to the membrane. Cholesterol oxidase is produced by some gram negative bacteria which can use cholesterol as a carbon source. Some of these organisms are pathogenic, such as Rhodococcus equi and Mycobacterium tuberculosis. It has been proposed that two surface residues, M332 and W333, contribute to binding by inserting their side chains into the hydrophobic core of the membrane. In order to test this hypothesis the following mutant enzymes will be made; M332A, M332C, W333A, W333C and W333F. The Cys mutants will be labeled with the fluorescent reporter groups acrylodan and monobromobimane. The mutant enzymes will be assayed for their ability to bind to vesicles of various lipid compositions under various conditions of ionic strength and pH. The mutants will also be tested for activity on cholesterol in micelles and in vesicles. The information gained from these studies will aid in improving serum- cholesterol assays, understanding the role of membrane cholesterol in receptor function and uncovering the role of cholesterol oxidase as a potential virulence factor.