Isolation and characterization of a monoclonal antibody against pig kidney sodium- and potassium-activated ATPase.
A hybridoma cell line producing mouse monoclonal antibody against pig kidney Na,K-ATPase was established. The antibody, named 38 (mAb38, IgG1), was purified from mouse ascites fluid by chromatography on a protein A-Sepharose column. Antigens immobilized on microplate wells with p-benzoquinone were used for titer assays. mAb38 cross-reacted with both dodecyloctaethyleneglycol monoether (C12E8)-solubilized enzyme and membranous sodium dodecyl sulfate (SDS)-treated enzyme from kidney with high affinity (50% binding = 0.6 nM). However, the antibody bound to neither alpha- nor beta-subunit separated by preparative SDS-polyacrylamide gel electrophoresis (PAGE). The stoichiometry of antibody binding to the purified enzyme was estimated to be about 0.86 mol of IgG per mol of alpha beta-protomer. Na,K-ATPase proteins were recovered from a column of mAb38-coupled Affi-Gel by elution with pH 3 buffer when C12E8-solubilized kidney enzyme or detergent extracts of brain microsomes were applied to it, confirming that the mAb is directed to Na,K-ATPase. mAb38 at saturation level concentrations had no effect on kidney Na,K-ATPase activity or on ouabain-sensitive Rb uptake in erythrocytes. In an immunofluorescence study, the antibody bound to intact erythrocytes much more strongly than control IgG1 (mAb50c), but the extent of the antibody binding to inside-out vesicles under hypotonic conditions was lower than that of the control. Most of the antibody binding activity remained when the kidney enzyme was treated with sialidase. These results suggest that this mAb38 was raised against an intact conformation of a cell-surface-exposed site of Na,K-ATPase.
O
O
Urayama
H
H
Nagamune
M
M
Nakao
Y
Y
Hara
H
H
Sugiyama
K
K
Sato
T
T
Nakao
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001921:Brain / Q000201:enzymology; D002846:Chromatography, Affinity; D000939:Epitopes; D006026:Glycoside Hydrolases; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology*; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D006026:Glycoside Hydrolases; D000254:Sodium-Potassium-Exchanging ATPase