A monoclonal antibody against a native conformation of the porcine renal Na+/K+-ATPase α-subunit protein

Abstract A monoclonal antibody (mAb50c) against the native porcine renal Na + /K + -transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na + /K + -ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to Na + /K + -ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na + , ATP or Mg 2+ (E1 form), slightly less in the presence of K + (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na + , Mg 2+ and ATP. The antibody inhibited both the Na + ,K + -ATPase activity and the K + -dependent p -nitrophenylphosphatase activity by 25%, but it had no effect on Na + -dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5′-isothiocyanatofluorescein after the addition of orthophosphate and Mg 2+ , and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the α-subunit.