Characteristics of antibody inhibition of rat kidney (Na+ -K+)-ATPase.

Antibodies which were raised against highly purified membrane-bound (Na+ -K+)-ATPase from the outer medulla of rat kidneys inhibit the (Na+-K+)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots. In the purified membrane-bound (Na+-K+)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na+ -K+)-ATPase activity (n = 0.86), (b) for the ATP binding to the enzyme (n = 0.58), and (c) for the ouabain inhibition of the (Na+ -K+)-ATPase activity (n = 0.77). By measuring the Na+ dependence of the (Na+ -K+)-ATPase reaction, a positive homotropic cooperativity (n = 1.67) was found. As reactivation of the antibody-inhibited enzyme proceeds very slowly (t0.5 = 5.2 hr), it was possible to measure characteristics of the antibody-(Na+ -K+)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na+ -K+)-ATPase reaction and on the ATP binding of the enzyme. Vmax of the (Na+ -K+)-ATPase reaction and the number of ATP binding sites were reduced while K0.5 ATP for the (Na+ -K+)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased the K0.5 value for the Na+ stimulation of the (Na+ -K+)-ATPase activity, but they did not alter the homotropic interactions between the Na+-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na+ -K)-ATPase activity was abolished by the antibodies. The data are tentatively explained by the following model: The antibodies bind to the (Na+ -K+)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na+ -K+)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na+ dependence of the (Na+ -K+)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.