A possible molecular mechanism of the action of digitalis: ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney.
Calcium binding at 0 degrees C to a purified sheep kidney Na+,K+-ATPase was described by linear Scatchard plots. Binding at saturating free calcium was 65-80 nmol/mg of protein, or 30-40 mol of calcium/mol of enzyme. Aqueous emulsions of lipids extracted from Na+,K+-ATPase yielded dissociation constants and maximum calcium-binding values that were similar to those for native Na+,K+-ATPase. Phospholipase A treatment markedly reduced calcium binding. Pretreatment of native Na+,K+-ATPase with ouabain increased the dissociation constant for calcium binding from 131 +/- 7 to 192 +/- 7 muM without altering maximum calcium binding. Ouabain pretreatment did not affect calcium binding to extracted phospholipids, ouabain-insensitive ATPases, or heat denatured Na+,K+-ATPase, Na+ and K+ (5-20 mM) increased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain increased the apparent affinity of binding sites for calcium. Extrapolation to physiological cation concentrations revealed that the ouabain-induced increase in apparent affinity for calcium may be as much as 2- to 3-fold. These results suggest: (1) calcium binds to phospholipids associated with Na+,K+-ATPase; (2) ouabain interaction with Na+,K+-ATPase induces a perturbation that is transmitted to adjacent phospholipids, altering their affinity for calcium; and (3) at physiological concentrations of Na+ or K+, or both, ouabain interaction with Na+,K+-ATPase may lead to an increased pool of membrane-bound calcium.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000327:Adsorption; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology*; D010740:Phospholipases / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011188:Potassium; D011239:Prednisolone / Q000494:pharmacology; D011489:Protein Denaturation; D012756:Sheep; D012964:Sodium / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D010743:Phospholipids; D010042:Ouabain; D007811:Lanthanum; D012964:Sodium; D011239:Prednisolone; D014700:Verapamil; D010740:Phospholipases; D000251:Adenosine Triphosphatases; D008274:Magnesium; D011188:Potassium; D002118:Calcium