KINETIC ANALYSIS OF THE MICROTUBULE NCD ATPASE

微管 NCD ATP 酶的动力学分析

• 批准号: 2701740
• 负责人: SUSAN P. GILBERT
• 金额: $ 18.99万
• 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/2701740
• 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.

该提案的长期目标是建立结构性和 一般生物马达产生力的机械基础和 特别是基于微管的 ATP 酶驱动蛋白和 ncd。 驱动蛋白是一种 微管依赖性运动负责细胞内运动 轴突中的膜细胞器。 它是第一个被表征的蛋白质 一类与多种驱动蛋白相关的 ATP 酶 生物过程。 驱动蛋白超家族的成员都共享 驱动蛋白运动域内具有显着的序列同源性，并且每个 驱动蛋白相关蛋白含有极高同源性的短片段 在 ATP 结合袋、微管结合域以及 以及 t=尚未在功能上定义的其他区域。 此外，每个 驱动蛋白相关蛋白含有一个独特的非运动结构域 据信赋予电机生物特异性。 Ncd 是一种驱动蛋白- 参与雌性减数分裂和胚胎有丝分裂的相关蛋白。 它是一个 与驱动蛋白相比，这是一个值得研究的有趣运动，因为有 40-45% 运动结构域约 350 个氨基酸内的序列同一性 意味着蛋白质将具有非常相似的三维空间 结构。 然而，作为马达，这两种 ATP 酶却截然不同。 Ncd 促进微管向相反方向易位 驱动蛋白，易位明显慢于驱动蛋白，并且 迄今为止的实验表明 ncd 并不是一种进行性 ATP 酶 驱动蛋白。 这里提出的研究的目的是了解 这些差异的机制基础。 具体目标是 1) 测量反应路径中每个步骤的动力学 microtubule-ncd ATPase，2) 确定 ncd ATPase 是否为 与微管的相互作用具有进行性或分布性。 到 实现这些目标需要详细而全面的动力 使用前稳态动力学技术进行热力学研究 包括快速淬火和停流方法。 仅凭此 直接测量酶活性位点的事件可以 获得机械信息以了解能量差异 由 ncd 和驱动蛋白 ATP 酶进行转导。