Immunochemical characterization of a functional site of (Na+,K+)-ATPase.
Several hybridoma cell lines secreting antibodies specific to the membrane (Na+,K+)-dependent ATPase from lamb kidney medulla have been isolated by using the methods developed by Kohler and Milstein. One of these antibodies (designated M7-PB- E9 ) has been shown to be directed against a functional epitope or antigenic site of the catalytic (alpha) subunit of the enzyme. Although this antibody was raised to the "native" holoenzyme, it has a higher apparent affinity toward the isolated, delipidated, and inactive alpha subunit than toward the holoenzyme. This antibody shows a 10-fold faster initial rate of binding to the alpha subunit than to the holoenzyme. The antibody dissociation rates from both isolated alpha subunit and holoenzyme are similarly slow, and the binding can be considered a pseudoirreversible reaction. By binding at this site, the antibody, however, acts like a "partial competitive inhibitor" with respect to ATP and acts as an uncompetitive or mixed competitive inhibitor with respect to the Na+ and K+ dependence of ATPase hydrolysis. This antibody also does not alter the cooperativity at either the Na+ or the K+ sites. The antibody causes a partial inhibition of the Na+- and MgATP-dependent phosphoenzyme intermediate formation but has no effect on either ADP in equilibrium ATP exchange or the K+-stimulated dephosphorylation step. In addition, the K+-dependent p-nitrophenylphosphatase activity of the enzyme was not affected. In the presence of Mg2+, the antibody stimulates the rate of cardiac glycoside binding [( 3H]ouabain) to the (Na+,K+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002460:Cell Line; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium