THE INHIBITORY MONOCLONAL-ANTIBODY M7-PB-E9 STABILIZES E(2) CONFORMATIONAL STATES OF NA+,K+-ATPASE

Monoclonal antibody M7-PB-E9 binds the sheep kidney Na+,K+-ATPase alpha-subunit with high affinity (K(d) = 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 (K(d) = 9 nM). In contrast, partial E2 conformations induced by KCl lowered eosin affinities (0.2 mM KCl, K(d) = 28 nM; 0.4 mM, K(d) = 86 nM), and M7-PB-E9 reduced these affinities further (K(d) = 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-PiMg2+, 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 a distinct from any ligand binding site and which plays an important role in the E1 reversible E2 transitions.