MEMBRANE BINDING OF CHOLESTEROL OXIDASE

胆固醇氧化酶的膜结合

• 批准号: 6385187
• 负责人: DAVID WOLFGANG
• 金额: $ 3.59万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-11 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6385187
• 关键词: acidity /alkalinity; active sites; binding sites; cholesterol; conformation; enzyme activity; enzyme mechanism; enzyme structure; fluorescent dye /probe; glycolipids; hydroxysteroid dehydrogenases; intermolecular interaction; ionic strengths; membrane biogenesis; membrane model; membrane structure; micelles; microorganism metabolism; molecular assembly /self assembly; site directed mutagenesis; structural biology

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.

本研究的目的是研究胆固醇氧化酶与膜的结合。 胆固醇氧化酶是由一些革兰氏阴性菌产生的，它们可以利用胆固醇作为碳源。 其中一些生物是致病性的，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，有助于通过将其侧链插入到膜的疏水核心结合。 为了检验这一假设，将制备以下突变酶：M332A、M332C、W333A、W333C和W333F。 Cys突变体将用荧光报告基团acrylodan和monobromobimane标记。 将测定突变酶在各种离子强度和pH条件下与各种脂质组合物的囊泡结合的能力。还将测试突变体对胶束和囊泡中胆固醇的活性。 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。