The objective of this proposal is to apply the techniques of molecular genetics to the analysis of structure function relationships for the Na,K-ATPase. We have developed an expression system to test the biological activity of cloned Na,K-ATPase genes. This system forms the framework for experiments designed to analyze the relationship between enzyme structure and function.
Specific aims i nclude: 1) Na,K-ATPase alpha/beta subunit interaction. We will attempt to determine which combinations of alpha and beta subunits can assemble to form holoenzyme. To address this issue, we will use epitope addition to tag a cDNA encoding a specific alpha and beta subunit isoform. Introduction of the construct into CV-1 cells and immunoprecipitation of the expressed fusion protein with an antibody against the epitope tag will allow us to determine which alpha/beta subunits are produced. This approach should also allow us to study isoenzyme function in transfected cells. 2) Structure-Function of the alpha subunit. We will attempt to identify sequences responsible for the variation in Na+ affinity between the alpha1 and alpha3 subunit isoforms. Construction and expression of chimeras between alpha1 and alpha3 subunit cDNAs should permit identification of sites within the alpha subunit that interact with Na+ and contribute to Na+ binding. A second approach will be designed to analyze the biochemical properties of the alpha2 isoform. These experiments should allow us to derive a clearer understanding of the functional relationships among the three alpha subunit isoforms. 3) Function of the beta subunit. To analyze the role of the beta subunit, we will take advantage of the fact that a cDNA encoding the beta subunit of the H,K-ATPase can confer ouabain resistance to primate cells by transfection. We will use epitope addition to determine if the H,K- ATPase beta subunit can assemble with the Na,K-ATPase alpha subunit. The construction and expression of chimeric H,K-/Na,K-ATPase beta subunit cDNAs should permit us to identify sites within the beta subunit that contribute to ouabain resistance. Expression of the beta subunit provides an opportunity to study structure function relationships by expressing beta subunits with alterations in primary amino acid sequence. These experiments also have practical significance because of the use of cardiac glycosides in the treatment of congestive heart failure.
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