KINETIC ANALYSIS OF THE MICROTUBULE NCD ATPASE

微管 NCD ATP 酶的动力学分析

• 批准号: 2415364
• 负责人: SUSAN P. GILBERT
• 金额: $ 18.73万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415364
• 关键词: SDS polyacrylamide gel electrophoresis; active sites; adenosinetriphosphatase; binding proteins; cell motility; chemical kinetics; conformation; gel filtration chromatography; hydrolysis; kinesin; meiosis; microtubule associated protein; protein structure function; sedimentation velocity; stoichiometry; thermodynamics; thin layer chromatography

The long term aims of this proposal are to establish the structural and mechanistic basis for force production by biological motors in general and the microtubule-based ATPases kinesin and ncd specifically. Kinesin is a microtubule dependent motor responsible for the intracellular movements of membranous organelles in axons. It is the first characterized protein of a class of kinesin-related ATPases implicated ina wide variety of biological processes. The members of the kinesin superfamily all share significant sequence homology within the kinesin motor domain, and each kinesin-related protein contains short segments of extremely high homology within the ATP binding pocket, the microtubule binding domain as well as well as t=other areas not yet defined functionally. In addition, each kinesin-related protein contains a nonmotor domain that is unique and is believed to confer biological specificity to the motor. Ncd is a kinesin- related protein involved in female meiosis and embryonic mitosis. It is an interesting motor to study in comparison to kinesin because there is 40-45% sequence identity within about 350 amino acids of the motor domain which implies that the proteins will have very similar three-dimensional structures. Yet as motors, the two ATPases are distinctively different. Ncd promotes microtubule translocations in the opposite direction of kinesin, translocations are significantly slower than kinesin's, and the experiments to date suggest that ncd is not a processive ATPase as is kinesin. The goal of the research proposed here is to understand the mechanistic basis of these differences. The specific aims are 1) to measure the kinetics of each step in the reaction pathway of the microtubule-ncd ATPase, 2) to determine whether the ncd ATPase is processive or distributive in its interactions with the microtubule. To accomplish these goals will require a detailed and comprehensive kinetic and thermodynamic investigation using presteady state kinetic techniques including rapid quench and stopped-flow approaches. It is only by this direct measurement of event at the active site of the enzyme can the mechanistic information be obtained to understand the differences in energy transduction by the ncd and kinesin ATPases.

这项建议的长期目标是建立结构性和