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， 神经细胞体与轴突末端的突触相连。 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质膜的运动。 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， 向外围）。 这种运动的能量来自于 水解三磷酸腺苷（ATP），纯化的驱动蛋白具有ATP酶 由微管刺激的活性。 本项目的目的是确定详细的酶机制 ATP的水解与运动有关。 将进行调查 以确定正向和反向的速率常数 对于水解方案中的基本步骤;即ATP的结合， 结合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。 速率常数为 在不存在微管的情况下以及作为 增加微管的水平。 还将进行平行研究 关于驱动蛋白与微管的物理相互作用的性质， 这在ATP水解过程中是如何变化的。 广泛使用将 由稳态动力学、同位素交换、 反应和光谱探针。 这些综合信息将 从这些研究中可以得到的信息将允许制定一个详细的 驱动蛋白诱导运动机制的模型及其在细胞内的作用 流程.