MECHANISM OF KINESIN ATPASE

驱动蛋白ATP酶的机制

• 批准号: 3415108
• 负责人: DAVID Daniel HACKNEY
• 金额: $ 10.26万
• 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 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3415108
• 关键词: Chlorophyta; adenosine diphosphate; adenosine triphosphate; adenosinetriphosphatase; conformation; enzyme mechanism; fluorescence spectrometry; fluorescent dye /probe; glyceraldehyde 3 phosphate dehydrogenase; high energy compound; intracellular transport; laboratory mouse; microtubule associated protein; microtubules; monoclonal antibody; neuronal transport; phosphates; radiotracer

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 eucaryotic 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 mechanisms of ATP hydrolysis is coupled to movement. Investigations will be conducted to determine the rate constants in both the forward and reverse directions for the elemental steps in the hydrolysis scheme; namely binding of ATP, hydrolysis of bound ATP, release of the bound products ADP and Pi, and any conformational changes which can be detected. The rate constants will be determined both in the absence of microtubules and as a function of increasing levels of microtubules. Parallel studies will also be performed on the nature of the physical interaction of kinesin with microtubules and how this changes during the process of ATP hydrolysis. Extensive use will be made of the techniques of steady state kinetics, isotopic exchange reactions, and spectroscopic probes. 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部位移动 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 所有桉树细胞。 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指示 某些类别的分泌囊泡向质膜运动。 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 （对应于神经细胞中的运动，远离核区域和 朝向外围）。 这种运动的能量是从 三磷酸腺苷的水解（ATP）和纯化的驱动蛋白具有ATPase 微管刺激的活性。 该项目的目的是确定详细的酶促机制 ATP水解的耦合与运动耦合。 将进行调查 确定向前和反向的速率常数 对于水解方案中的元素步骤；即ATP的结合， 绑定ATP的水解，结合产物ADP和PI的释放以及任何 可以检测到的构象变化。 费率常数将是 在没有微管的情况下确定 微管的水平增加。 也将进行平行研究 关于驱动蛋白与微管的物理相互作用的性质 在ATP水解过程中，这种情况如何变化。 广泛使用 由稳态动力学，同位素交换的技术制成 反应和光谱探针。 合并的信息将 从这些研究中获得，将允许制定详细的 驱动蛋白诱导的运动机制及其在细胞中的作用的模型 过程。