Isolation and characterization of monoclonal antibodies to (Na+ + K+)-ATPase.
Four stable hybridoma cell lines secreting antibodies specific to the membrane (Na+ + K+)-dependent ATPase isolated from lamb kidney medulla have been produced by fusing mouse myeloma cells with spleen cells from immunized mice. These cell lines produce IgG gamma 1 heavy chain and kappa light chain antibodies which are directed against the catalytic or alpha-subunit of the (Na+ + K+)-ATPase enzyme. Binding studies, using antibodies that were produced by growing hybridomas in vivo and purified by affinity column chromatography, suggest a somewhat higher affinity of these antibodies for the isolated alpha-subunit than for the 'native' holoenzyme. In addition, these monoclonal antibodies show no reactivity with either the glycoprotein (beta) subunit of the lamb enzyme nor the (Na+ + K+)-ATPase from rat kidney, an ouabain-insensitive organ. Cotitration binding experiments have shown that the antibodies from two cell lines originally isolated independently from the same culture plate well population of fused cells bind to the same determinant site and are probably the same antibody. Cotitration and competition binding studies with two other antibodies have revealed two additional distinct antibody binding sites which appear to have little overlap with the first site. One of the three different antibodies isolated caused a partial inhibition of the (Na+ + K+)-ATPase activity. This antibody appears to be directed against a specific functionally important site of the alpha-subunit and is a competitive inhibitor of ATP binding. Under optimum conditions of ATPase activity, this inhibitory effect is not altered by the presence of the other two antibodies.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D006825:Hybridomas / Q000276:immunology; D007679:Kidney Medulla / Q000201:enzymology*; D008214:Lymphocytes / Q000276:immunology; D046911:Macromolecular Substances; D051379:Mice; D008815:Mice, Inbred Strains; D010954:Plasmacytoma / Q000276:immunology; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D000254:Sodium-Potassium-Exchanging ATPase