CELL & MOLECULAR BIOLOGY OF (NA+ + K+) ATPASE

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The (Na+ and K+) ATPase is an oligomeric plasma membrane protein complex which catalyzes the ATP-dependent exchange of Na+ and K+ ions across the plasma membrane. Recent experimental evidence suggests that this enzyme exists as several distinct isoforms in mammalian tissues. In order to characterize the structure-function relationships of (Na+ and K+) ATPase isoforms, we will isolate and sequence full length complementary DNA (cDNA) clones of the alpha (catalytic) and beta subunits of rat (Na+ and K+) ATPase. Based upon the deduced amino acid sequences from these clones, site specific, affinity purified polyclonal antibodies will be raised against appropriate synthetic peptides and/or fusion proteins. These antibodies will be utilized to characterize protein expression, targeting, and processing in cultured rat cells and tissues by immunoprecipitation of metabolically labelled proteins, immunofluorescence, colloidal gold immunocytochemistry, and immunoblot analysis. In vitro transcription vectors will be constructed from full length alpha and beta subunit cDNA clones. RNA from in vitro transcription of these vectors will be translated in wheat germ and/or reticulocyte lysate microsomal membrane systems. We will characterize membrane insertion, protein topology, and post- translational modification of each subunit, as well as subunit association and functions of the (Na+ and K+) ATPase. We will evaluate (Na+ and K+) ATPase expression, targeting, and function in heterologous eukaryotic cells transfected with eukaryotic expression vectors containing full length rat alpha and/or beta subunit cDNAs. Site directed mutagenesis and gene fusion strategies will be utilized to extend in vitro translation and cell expression approaches. We will seek to directly examine how changes in subunit primary structure lead to changes in enzyme targeting, processing, subunit association, and function. These complementary approaches will combine molecular biology, protein biochemistry, and cell biology to further elucidate the function of the (Na+ and K+) ATPase family of proteins. These studies will lead to a better understanding of the biology of (Na+ and K+) ATPase in normal and disease states, both in epithelial and excitable tissues (such as nerve or muscle). This enzyme provides an excellent model for studying the targeting, processing, and function of polarized plasma membrane proteins.