LOCALIZATION OF LIGAND INTERACTIONS WITH THE SODIUM PUMP

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The purpose of this work is to identify the regions and sites on the sodium pump protein where physiological ligands interact and which are essential for the mechanism of active sodium transport. Experimental studies over the past three decades have established the relationships between the biochemical reactions catalyzed by the sodium pump and the transport reactions it mediates. An approach directed at the identification of such regions is particularly timely as the primary sequence of the alpha and beta subunits has recently been obtained. The present work will utilize purified sodium pump protein from canine renal medulla and will employ photochemical and chemical affinity labeling of specific regions of the protein. The kinetics and mode of inhibition of a variety of novel reagents will be characterized and their sites of action localized using radiolabels. The radiolabeled protein will be subjected to proteolysis and labeled peptides isolated prior to partial sequencing and localization in the primary structure. The reagents to be used include analogs of a new carboxyl-selective fluorescent reagent which has recently been shown to be a K-site label, an analog of a well known diuretic, amiloride, which recent work has shown may also be a probe for the K-site. Photosensitive reagents will also be used to localize the region near an essential arginine in the ATP binding domain of the alpha-subunit. Also, further use will be made of a photoaffinity reagent, NAB-ouabain, which binds specifically to the cardiac glycoside site on the outside surface of the Na,K-ATPase, to identify the amino acids which are close to the glycoside moiety in the ouabain binding domain. The overall aim is to determine where on the primary sequence the residues are which interact with physiological ligands and which residues are involved in essential conformational transitions in the sodium pump transport cycle. The sodium pump is vital in a variety of organs for fluid and electrolyte balance. These processes are disturbed in a variety of disease states. Before an adequate description of these pathological situations can be made a more complete understanding of the functioning of the sodium pump is required.