The long-term goal of this research is to understand how ATP drives ion transport via the Na,K-Atpase. A great deal of evidence supports a mechanism in which ATP hydrolysis is linked to the transport steps through conformational transitions induced by substrate and ions. Despite the fact that the enzymatic properties are known in considerable detail, little is known about the structures of the major E1 and E2 states of the Na,K-ATPase, and even less about proposed subconformations. This project consists of experiments designed to delineate further the conformational states of the enzyme, in particular the interconversions and the structural and functional relationships among them. Current studies are continued and extended in five major areas of structural and kinetic investigations. (1) Determination of structural relationships among enzyme states formed by conformationally selective treatments using distances measured by Forster energy transfer. (2) Measurement of rates of the conformational steps in the reaction cycle with fluorescence stopped-flow. (3) Labeling of the putative ion binding domain with fluorescent carbodiimides. (4) Determination of the function of the IAF binding region using antibodies raised to a purified peptide. (5) Investigation of species differences in fluorescent labeling of Na,K-ATPase by protein sequencing techniques. Ion transport across membranes is precisely regulated by specific transport systems. There is a "pump" protein which exchanges sodium ions and potassium ions across membranes. This plays an important role in regulating cell volume and acidity and ion composition. It is known that the pump protein changes shape during ion transport. It is important to the cell to conserve energy during this process. This project will provide new information on the structure of the pump and its involvement in energy metabolism.*** //
