The (Na+,K+)-activated adenosine triphosphatase [(Na,K)-ATPase] is the molecular machine which effects the coupled transports of Na+ and K+ in all animal cells. To understand the molecular mechanism of this machine, its three-dimensional structure must be elucidated. Among other things, this requires a knowledge of the amino acid sequences of the subunits (Alpha and Beta). Because of large stretches of hydrophobic amino acids which cause aggregation of partial hydrolytic products, attempts to sequence the protein over a period of more than five years by classical amino acid sequencing have failed. The primary objective of the proposed research is to determine the amino acid sequence of the subunits by preparation of cDNA clones, determination of the nucleotide sequences of the cDNA inserts, and deriving the amino acid sequences from the nucleotide sequences. Another objective is to study the site of synthesis and post-translational modifications of the subunits. The site of synthesis will be determined by translation of mRNA from free and membrane-bound polysomes. The post-translational modifications (cleavage and glycosylation) will be examined in a cell-free translation system containing microsomes from dog pancreas. We will also study developmental regulation of the synthesis of the (Na,K)-ATPase subunits in the brine shrimp, Artemia salina, by examining protein synthesis and specific mRNA levels. The levels of mRNA will be estimated by hybridization analysis and quantitation of specific cell-free translation products at various times throughout brine shrimp development. Processing (proteolytic cleavage and glycosylation) will also be studied, as well as the mechanism of insertion into the membrane.
