Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study.
A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology*; D004285:Dogs; D000939:Epitopes / Q000276:immunology; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007159:Immunologic Tests; D007679:Kidney Medulla / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D014357:Trypsin; D010206:Papain; D000254:Sodium-Potassium-Exchanging ATPase