Studies of the antigenic properties of the catalytic and glycoprotein subunits of Na+,K+-ATPase.
Antibodies were raised against isolated, delipidated catalytic [alpha] and glycoprotein [beta] subunits of the Na+,K+-dependent ATPase purified from lamb kidney medulla. The specificity of each antiserum was confirmed by agar double-diffusion precipitation, immunoelectrophoresis, and polyacrylamide gel electrophoresis. A solid phase adsorption assay was also employed to determine antibody binding titers and to further test the specificity of these antisera. Antibodies raised to the alpha subunit had a strong reactivity and similar titer values for both the holoenzyme and the alpha subunit and a low-affinity cross-reactivity with the beta subunit. In contrast, beta-subunit-directed antibodies had little reactivity or binding with the holoenzyme and a low-affinity cross-reactivity with the alpha subunit. Competition binding studies revealed that about 80% of the alpha-subunit-specific antibodies bound to the holoenzyme, indicating that similar sets of antigenic sites are exposed in the lipid-embedded holoenzyme complex and in the isolated alpha subunit. Competition binding studies also suggest that the subunit cross-reactivities of the antisera may not result from simple contamination of the respective antigens, but that there may be partial homologies of some antigenic sites. In addition, the beta-directed antibodies had no effect on Na+,K+-ATPase activity, while the alpha-directed antibodies were effective inhibitors of activity. This indicates that at least some functionally important antigenic sites of the alpha subunit may be unaltered by its isolation and delipidation.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D000918:Antibody Specificity; D000941:Antigens / Q000302:isolation & purification*; D001666:Binding Sites, Antibody; D002384:Catalysis; D006023:Glycoproteins / Q000276:immunology*; D007679:Kidney Medulla / Q000201:enzymology; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology*
D000906:Antibodies; D000941:Antigens; D006023:Glycoproteins; D000254:Sodium-Potassium-Exchanging ATPase