TY - JOUR T1 - The Role of Phospholipids in the Binding of Ouabain to Sodium- and Potassium-Dependent Adenosine Triphosphatase JF - Molecular Pharmacology JO - Mol Pharmacol SP - 350 LP - 359 VL - 9 IS - 3 AU - KAZUYA TANIGUCHI AU - SHOICHI IIDA Y1 - 1973/05/01 UR - http://molpharm.aspetjournals.org/content/9/3/350.abstract N2 - The effect of treatment with phospholipase A (phosphatide acylhydrolase, EC 3.1.1.4) on the binding of ouabain to (Na+ + K+)-ATPase (ATP phospholydrolase, EC 3.6.1.3) as well as on (N+ + K+)-ATPase activity was studied in the presence of various concentrations of ATP and other ligands. The time courses of [3H]ouabain binding to ATPase in response to phospholipase A were the same in the presence of Mg2+ + ATP and of Mg2+ + Na+ + ATP as under control conditions. However, after phospholipase treatment the initial rates of ouabain binding were reduced approximately to 40% of the control in the presence of Mg2+ + K+ + ATP, Mg2+ + Pi, or Mg2+. In the presence of both phosphatidylserine and phosphatidylinositol, the ouabain-binding rate increased significantly. The maximum amounts of ouabain binding in the presence of various ligands and 1-250 µM [3H]ouabain were used to estimate binding capacities and dissociation constants for ouabain. The binding capacities of the ouabain-binding site showed no remarkable change as a consequence of phospholipase A treatment. ATPase preparations treated either with or without phospholipase A apparently had two different kinds of ouabain-binding sites in the presence of various physiological ligands. The values for the dissociation constant of the low-affinity site were approximately 10-100 times those of the high-affinity site in the presence of various ligands. The values for the dissociation constant of the high-affinity site in the presence of Mg2+ + ATP and Mg2+ + Na+ + ATP were not changed by phospholiase A treatment. The apparent dissociation constant of the ouabain high-affinity site in the presence of Mg2+ + K+ + ATP, g2+ + Pi, or Mg2+ was increased by the phospholipase A treatment. The ligands used reduced the binding affinities of both the high- and low-affinity sites of the control preparation as follows: Mg2+ + Pi ≈ Mg2+ + Na+ + ATP ≈ Mg2+ + ATP, Mg2+ + Na+ + K+ + ATP, Mg2+ + K + ATP ≈ Mg2+. The number of high-affinity sites in the presence of Mg2+ + Pi appeared to be greater than in the presence of other ligands. The number of low-affinity sites increased 2-3-fold in the presence of K+ as compared to its absence. The number of sites phosphorylated on the enzyme in the presence of Mg2+ + Na+ + ATP was approximately the same as the number of high-affinity ouabain-binding sites. The Vmax values with the high and low concentrations of ATP were reduced 70% by phospholipase A treatment. The apparent Km at low concentrations of ATP was decreased 50%. These results suggest that the enzyme preparation contains 1 mole each of high- and lowaffinity ouabain-binding sites per mole of phosphorylated sites. The reactivity of the highaffinity site with ouabain in the presence of Mg2+ + K+ + ATP, Mg2+ + Pi, and Mg2+, but not Mg2+ + ATP or Mg2+ + Na+ + ATP, was changed by phospholipase A treatment. This indicates that there are at least tow kinds of ouabain-binding conformations, one induced by Mg2+ + K+ + ATP, Mg2+ + Pi, or Mg2+ in which phospholipids play a role, and another, found in the presence of Mg2+ + ATP or of Mg2+ + Na+ + ATP, in which phospholipids do not play a role. ACKNOWLEDGMENTS We wish to express our thanks to Dr. D. Foster, Department of Physiology, Vanderbilt University, and to Dr. F. Morita, Department of Chemistry, Hokkaido University, for their helpful advice and valuable discussons. We are also indebted to Mr. E. Kasuga for his technical assistance. ER -