Abstract
pH influences the equilibrium binding and the association and dissociation rate constants (ka and kd) of ouabain and digoxigenin for (Na+ + K+)-ATPase. When the cardiotonic steroid-enzyme complexes are formed in the presence of Mg2+ and Pi (type II complex) between pH 6.5 and 8.5, the ka values of ouabain and digoxigenin and the apparent number of sites according to Scatchard plots of digoxigenin binding are reduced as the pH is increased. These changes correspond to changes in enzyme phosphorylation by Mg2+ and Pi. The kd values of type II complexes with ouabain and digoxigenin are constant below pH 8.0, but they decrease significantly to a new plateau at about pH 8.0 and above. This change is slow and reversible, and it is produced only by changing the pH of the dissociation medium. In the cardiotonic steroid-enzyme complex formed in the presence of Na+, Mg2+, and ATP (type I complex), the ka values of ouabain, the apparent number of sites for digoxigenin binding, and enzyme phosphorylation by ATP are not influenced by pH. On the other hand, the kd of the type I ouabain-enzyme complex decreases with increasing pH; the kd at pH 8.0 is one-fifth the value at pH 6.5. The kd of the type I ouabain-enzyme complex is not dependent on pH during complex formation but is dependent on pH during dissociation. These effects of pH on the ka values of both cardiac glycoside and steroid support the reaction mechanism previously proposed: that cardiac steroids on glycosides bind to the phosphorylated form of (Na+ + K+)-ATPase and that binding of the steroid portion results in activation of the sugar-binding site, with consequent binding of the glycoside portion.
- Copyright © 1978 by Academic Press, Inc.
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