STRUCTURAL ANALYSIS OF THE DYNEIN ATPASE

动力蛋白ATP酶的结构分析

• 批准号: 3280577
• 负责人: KENNETH ALLEN JOHNSON
• 金额: $ 9.97万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1986-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280577
• 关键词: adenosinetriphosphatase; cilium /flagellum motility; cytoskeleton; electron microscopy; enzyme linked immunosorbent assay; enzyme structure; gel electrophoresis; ion exchange chromatography; monoclonal antibody; proteolysis; scanning electron microscopy

Previous work in this laboratory by scanning transmission electron microscopy (STEM) has shown that Tetrahymena 21S dynein consist of a bouquet of three globular heads connected by three separate strands to a common base. Mass analysis by integration of electron scattering intensities gave a molecular weight of 2x10 to the 6 g/mole and indicated that two of the globular heads were identical with a mass of approximately 400 kdaltons while the third head exhibited a mass of approximately 550 kdaltons. Moreover, the present data suggests that the rootlike base of the bouquet forms the structural attachment site and the globular heads interact with the B-subfiber in an ATP-dependent reaction coupled to force production. Specifically, the current proposal seeks to follow up on these studies to (1) establish the absolute orientation of the molecule with respect to the A-subfiber of the outer doublet. (2) Determine whether two heads are identical and one head is different. (3) Localize the ATP and microtubule binding sites. (4) Localize the major polypeptides of the dynein molecule. (5) Begin to address the question of the roles of the three dynein heads in motility. These problems will be addressed by STEM and conventional TEM analysis of isolated dynein, intact ciliary outer doublets and dynein subfragments produced by proteolytic digestion. Monoclonal antibodies will be used to localize functional domains of the dynein molecule. This structural analysis is absolutely essential to the longer term objectives of establishing the mechanism of force pro-production and control of the dynein in ciliary motility and for subsequent analysis of dynein-like ATPases in other microtubule-dependent movements.

本实验室以前的工作是通过扫描透射电子进行的