Location of major antibody binding domains on alpha-subunit of dog kidney Na+-K+-ATPase.

The locations of binding sites on the alpha-subunit of dog kidney Na+-K+-ATPase for both monoclonal antibodies and antibodies from polyclonal antisera have been determined. Three distinct regions of the alpha-subunit, all located within the amino terminal half of the polypeptide, were recognized by the antibodies: a region near the amino terminus of the polypeptide and two regions that are separated by a site for trypsin cleavage of the ATPase in KCl. No significant binding of antibodies to the carboxy terminal region of the alpha-subunit was detected. The binding sites for the antibodies are located within regions of the polypeptide predicted to be exposed within the cytoplasm of the cell (P. L. Jorgensen, S. J. D. Karlish, and C. Gitler, J. Biol. Chem. 257: 7435-7442, 1982). This prediction was verified by the demonstration that the antibodies did not react with Na+-K+-ATPase in tight right-side-out vesicles but would bind to the protein after the vesicles had been disrupted with detergent. A model for the folding of the alpha-subunit through the membrane, based on these data, is presented.