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 from plasma membrane and the Ca2+-ATPase from sarcoplasmic reticulum, primarily by determining the detailed locations, geometries and interactions of the various substrates, activators and inhibitors on these enzyme transport systems. The conformations of bound ATP on these ATPases will be determined by two distinct techniques (nuclear relaxation with paramagnetic probes, and also transferred nuclear Overhauser enhancement (TRNOE) measurements. A new ouabain spin label prepared in this laboratory will be used to examine the ouabain binding site and the extracellular surface of the (Na+ + K+)-ATPase. This label will be observed directly by conventional EPR and also by saturation transfer methods, and Li-7 NMR studies will examine the interactions of the ouabain site with potassium sites on the extracellular surface of the enzyme. Proton NMR studies of the Ca2+-ATPase will be directed to the identification of aromatic amino acids in the vicinity of the Ca2+ binding sites using lanthanide shift reagents such as Yb3+ and the diamagnetic lanthanides, La3+ and Lu3+, to complement ongoing studies with Gd3+, a paramagnetic broadening agent and EPR probe which binds with high affinity to two calcium binding sites on the enzyme. Spectroscopic data for the C12E8-solubilized ATPases will be compared with data from the native enzyme to assess the possible involvement of subunit-subunit interactions in these systems. The relationship between functional domains in the alpha subunits of these enzymes will be examined using tryptically and chymotryptically cleaved preparations of the enzymes.
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