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.
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