MECHANISM OF KINESIN ATPASE

驱动蛋白ATP酶的机制

• 批准号: 2267005
• 负责人: DAVID Daniel HACKNEY
• 金额: $ 21.03万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2267005
• 关键词: adenosine triphosphate; adenosinetriphosphatase; cell motility; conformation; enzyme inhibitors; enzyme mechanism; enzyme structure; high energy compound; hydrolysis; intracellular transport; kinesin; microtubules; neuronal transport; protein isoforms; proteolysis; synthetic peptide

The long range goal of this research is to provide a better understanding of the mechanism of movement of intracellular organelles along microtubules. Such movement plays a special role in the process of fast axonal transport in nerve cells. This process provides one means for the movement of newly synthesized materials from their site of synthesis in the body of a nerve cell to the synapse at the end of the axon. Similar motility processes, however, also are likely to play an important roles in all eukaryotic cells. For example, the directed movement of membranous organelles has been implicated in the extension of the endoplasmic reticulum and mitochondria away from the nuclear region and in the directed movement of some classes of secretory vesicles towards the plasma membrane. The protein kinesin has recently been isolated and shown to be a motor for driving movement along microtubules in the anterograde direction (corresponding to movement in a nerve cell away from the nuclear region and toward the periphery). The energy for this movement is derived from hydrolysis of adenosine triphosphate (ATP), and purified kinesin has ATPase activity which is stimulated by microtubules. The aim of this project is to determine the detailed enzymatic mechanism of ATP hydrolysis by the kinesin/microtubule motility system with emphasis on how hydrolysis is coupled to movement. Recent progress indicates that kinesin undergoes a large conformational change as a function of ionic strength and a major component for the upcoming grant period is the further characterization of this conformational change and its possible significance for the regulation of kinesin. An important approach will be the characterization of individual domains of kinesin and their interaction. These domains will be obtained by limited proteolysis and by expression of fragments of kinesin cDNA. Resolution of these issues will allow a more detailed kinetic analysis to be undertaken on the forms of kinesin which are properly activated. Extensive use will be made of steady state and single turnover kinetics and immunological approaches. The combined information which will be available from these studies will allow the formulation of a detailed model for mechanism of motility induced by kinesin and its role in cellular processes.

这项研究的远距离目标是提供更好的理解 细胞内细胞器运动机理 微管。这种运动在快速过程中起着特殊的作用 神经细胞中的轴突运输。这个过程为 新合成材料从其合成位置移动 神经细胞的身体在轴突末端到突触。相似的 但是，运动过程也可能在 所有真核细胞。例如，膜的定向运动 细胞器与内质的扩展有关 网状和线粒体远离核区域和 某些类别的分泌囊泡向等离子体的定向运动 膜。 蛋白驱动蛋白最近被分离出来，并显示为 沿着微管沿着微管驱动运动 （对应于远离核区域的神经细胞中的运动 朝向外围）。这种运动的能量是从 三磷酸腺苷的水解（ATP）和纯化的驱动蛋白的水解 由微管刺激的ATPase活性。 该项目的目的是确定详细的酶促机制 驱动蛋白/微管运动系统的ATP水解系统 关于如何将水解与运动耦合。最近的进展表明 运动蛋白经历了大构象变化作为离子的函数 力量和即将到来的赠款期的主要组成部分是 这种构象变化及其可能的进一步表征 调节运动蛋白的重要性。一个重要的方法将是 动力蛋白及其单个领域的表征 相互作用。这些域将通过有限的蛋白水解和 驱动蛋白cDNA片段的表达。解决这些问题将 允许对以下形式进行更详细的动力学分析 适当激活的动力蛋白。广泛使用将是稳定的 状态和单个失误动力学和免疫学方法。这 这些研究将获得的组合信息将允许 由详细模型制定了由 动力素及其在细胞过程中的作用。