The Na pump is responsible for maintaining the intracellular composition of Na and K ions in most eukaryotic cells. This protein is composed of alpha and beta-subunits which have both been recently cloned. The alpha- subunit (mol. wt. 112 kD) is composed of 8 to 10 transmembrane helices and a larger cytoplasmic loop. This loop contains all of the amino acids which have been identifiable as essential for ATP binding or phosphorylation. There is significant homology among ion pumps or P-type ATPases and recent models based on homologies with other ATP-binding proteins model this domain in a series of alpha-helices and beta-sheets. The present work will take advantage of the techniques of molecular physiology to obtain more detailed structural information on this domain, which has recently been obtained in isolation from an over-expression system in bacteria. The protein binds ATP and the present work proposes to investigate the properties of this isolated loop and obtain information on its structure. In other studies, membranes from baculovirus infected Sf9 cells will more closely define its role in the overall cycle. Recent studies from the applicant's laboratory have demonstrated a novel form of post-transnational processing of the alpha- subunit, a novel N-glycosylation. It is proposed to further characterize the sites of glycosylation. The beta-subunit or glycoprotein of the Na pump has until now, not been identified with a essential role in catalysis or transport. We have shown that the beta-subunit is essential for activity. Reduction of S-S bridge in he extracellular domain abolishes cation occlusion. The present work proposes to investigate further this essential role for the beta-subunit and to characterize its role in Na and K activated further ATP hydrolysis and transport. The Na pump is vital in kidney and intestine for fluid electrolyte balance; it is also an essential component in the maintenance of membrane excitability and cardiac function. Many of these processes are disturbed in pathological states. Before an understanding of these disease states can be achieved, a more complete understanding of structure-function relations in the normally functioning Na pump is required. The proposed studies will contribute to a greater understanding of this very important active transport system.
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