The primary goal of this research is to determine the mechanism of ATP hydrolysis and ion transport for several membrane-bound, ion transport ATPases, the (Na+ + K+) ATPase and Mg2+ AtPase from plasma membrane and the Ca2+ AtPase from sarcoplasmic reticulum, primarily be determining the detailed locations, geometries and interactions of the various substrates, activators and inhibitors on these enzyme transport systems. These enzymes, which play important roles in many metabolic processes, including muscle contraction and excitability in brain and nerve tissue, will be studied using a variety of nuclear magnetic resonance, electron paramagnetic resonance and kinetic studies. The active sites of these enzymes will be characterized with respect to substrate and metal ion binding, and paramagnetic probes such as Mn2+, Gd3+, Cr3+ and nitroxide spin probes will be used as probes to determine the conformations and locations of ion and substrate binding sites on the enzyme. These prurified enzymes will be reconstituted into lipid vesicles and studied by NMR, EPR and kinetic methods in order to characterize the orientation of the enzymes in the membrane, the interactions of subunits in the membrane, and the mechanism of ion transport. In these studies, we will use several techniques which we have developed for use with these enzymes, including Gd3+ EPR and lithium NMR methods.
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