A monoclonal antibody against a native conformation of the porcine renal Na+/K(+)-ATPase alpha-subunit protein.
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 Mg2+ (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+, Mg2+ 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 Mg2+, 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 alpha-subunit.
Urayama
O
O
Department of Biochemistry, Tokyo Medical and Dental University School of Medicine, Japan.
Nagamune
H
H
Nakao
M
M
Hara
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine; D013861:Thiocyanates
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008024:Ligands; D046911:Macromolecular Substances; D010446:Peptide Fragments; D013861:Thiocyanates; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate