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.

这项研究的长期目标是提供一个更好的理解 细胞内细胞器沿着运动的机制 微管这种运动在快速的过程中起着特殊的作用 神经细胞中的轴突运输。这一过程提供了一种手段， 新合成的材料从它们的合成位置移动， 从神经细胞体到轴突末端的突触。类似 然而，运动过程也可能在以下方面发挥重要作用： 所有的真核细胞。例如，膜的定向运动 细胞器与内质网的延伸有关， 网状细胞和线粒体远离核区， 某些种类的分泌囊泡向血浆的定向运动 膜的 蛋白质驱动蛋白最近已被分离出来，并显示出是一个马达， 在顺行方向上沿沿着微管驱动运动 （对应于神经细胞远离核区域的运动） 并朝向外围）。这种运动的能量来自于 三磷酸腺苷（ATP）的水解和纯化的驱动蛋白具有 由微管刺激的ATP酶活性。 本项目的目的是确定详细的酶促机制 ATP水解的驱动蛋白/微管运动系统，重点 水解与运动的联系最近的进展表明， 驱动蛋白作为离子的函数经历大的构象变化， 的实力和即将到来的赠款期的一个主要组成部分是 进一步表征这种构象变化及其可能的 对驱动蛋白调控的意义。一个重要的方法是 驱动蛋白的各个结构域的表征及其 互动这些结构域将通过有限的蛋白酶解和 驱动蛋白cDNA片段的表达。这些问题的解决将 允许对以下形式进行更详细的动力学分析： 驱动蛋白被适当激活。将广泛使用稳定的 状态和单周转动力学和免疫学方法。的 从这些研究中获得的综合信息将允许 的运动机制的详细模型的制定， 驱动蛋白及其在细胞过程中的作用。