The enzyme (Na,K)ATPase, also known as the sodium pump, is a protein that is found embedded within the plasma membranes of nearly all animal cells. The function of the sodium pump is to transport sodium out of the cell, and to transport potassium into the cell, against the existing concentration gradients for these ions. The energy for the performance of this work is derived from the hydrolysis of ATP the protein. The (Na,K)ATPase is the only known receptor for digitalis-like drugs, and by regulating the intracellular concentration of sodium the pump is involved in the electrical activity of nerve and muscle, the volume regulation of most cells, the transport of many substrates across epithelial cells, and possibly in the response of cells to certain hormones and growth factors. Despite the important role of the sodium pump in physiology, the mechanism of active transport catalyzed by this protein in unknown. As one aspect of the study of this problem, this application describes experiments designed to obtain structural data about this protein. Photochemical probes for the active site of ATP hydrolysis and for the cardiac glycoside binding site will be synthesized and used to identify and purify peptides derived from these sites. The amino acid sequences of these peptides will be determined. In addition, peptides from other locations within the protein will be purified and sequenced, and the sequences of all of these peptides will be examined for the most appropriate sequences for the design of synthetic oligonucleotide probes. These probes will be prepared and used to screen a cDNA library made from size-fractionated dog kidney mRNA in order to identify recombinants that contain sequences corresponding to parts of the (Na,K)ATPase gene. The nucleic acid will be sequenced and from it the protein sequence will be inferred. The sequences corresponding to the peptides obtained from the active site and from the ouabain binding site will be identified within the whole sequence. Other sequences from membrane-embedded regions of the protein, as well as sequences from the functional sites, will also be identified. With this information and the predictions of computer models for protein structure it may be possible to deduce the structural features of the protein that are involved in the active transport of sodium and potassium.
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