The inhibitory monoclonal antibody M7-PB-E9 stabilizes E2 conformational states of Na+,K(+)-ATPase.
Monoclonal antibody M7-PB-E9 binds the sheep kidney Na+,K(+)-ATPase alpha-subunit with high affinity (Kd = 3 nM) and inhibits enzyme turnover in competition with ATP, and, like ATP, in the presence of Mg2+, it stimulates the rate of ouabain binding [Ball, W. J. (1984) Biochemistry 23, 2275-2281]. In this study, covalent attachment of fluorescein 5'-isothiocyanate (FITC) at (or near) the enzyme's ATP binding site did not alter the antibody's affinity for alpha nor did bound antibody alter the anisotropy of (r = 0.36) or the solvent accessibility of iodide to bound FITC. Further, in its E1Na+ conformation (4 mM NaCl), the enzyme's affinity for the ATP congener eosin was unaltered by the bound antibody (Kd = 9 nM). In contrast, partial E2 conformations induced by KCl lowered eosin affinities (0.2 mM KCl, Kd = 28 nM; 0.4 mM, Kd = 86 nM), and M7-PB-E9 reduced these affinities further (Kd = 66 and 130 nM, respectively). By monitoring the fluorescence changes of the FITC-labeled enzyme, the antibody was found to assist several ligand-induced conformational transitions from E1 (E1Na+ or E1Tris) to E2 (E2K+, E2-P(i)Mg2+, or E2Mg2+.ouabain) states, and inhibit the E2K(+)-->E1Na+ transition. Antibody binding alone, however, did not appear to significantly alter enzyme conformation. The antibody therefore is not directed against the ATP site but binds to a region of alpha distinct from any ligand binding site and which plays an important role in the E1<-->E2 transitions.
Abbott
A
A
Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Ohio 45267-0575.
Ball
W J
WJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D016650:Fluorescein-5-isothiocyanate; D007679:Kidney Medulla / Q000201:enzymology; D010042:Ouabain / Q000378:metabolism; D011487:Protein Conformation; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate