MECHANISM OF KINESIN ATPASE

这项研究的长期目标是提供一个更好的理解 细胞内细胞器的运动机制 微管。这种运动在斋戒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方法 新合成材料在其合成位置的移动 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，并在定向 某些类型的分泌囊泡向质膜移动。 该蛋白最近被分离出来，并被证明是一种运动 沿微管顺行方向驱动运动 (对应于神经细胞离开核区的运动和 朝向外围)。这种运动的能量来源于 三磷酸腺苷(ATP)的水解酶和纯化的Kinesin具有ATPase 由微管刺激的活动。 这个项目的目的是确定详细的酶作用机制。 三磷酸腺苷的水解度与运动相联系。将进行调查 确定正向和反向的速率常数 对于水解方案中的基本步骤；即结合ATP， 结合的ATP的水解，结合的产物ADP和PI的释放，以及任何 可以检测到的构象变化。速率常数将为 在没有微管的情况下确定的，以及作为 微管水平增加。还将进行平行研究。 关于激动素与微管和微管相互作用的性质 在三磷酸腺苷的水解过程中这种变化是怎样的。广泛使用将 由稳态动力学、同位素交换等技术组成 反应和光谱探测器。合并后的信息将 从这些研究中得到的将允许制定一份详细的 激动素诱导运动机制的模型及其在细胞中的作用 流程。

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.