Molecular Pharmacology, Vol 9, 350-359, Copyright © 1973 by the American Society for Pharmacology and Experimental Therapeutics

The Role of Phospholipids in the Binding of Ouabain to Sodium- and Potassium-Dependent Adenosine Triphosphatase

¹ Department of Pharmacology, School of Dentistry, Hokkaido University, Sapporo, Japan

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⁺ + P_i, 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⁺ + P_i, 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⁺ + P_i Mg²⁺ + Na⁺ + ATP Mg²⁺ + ATP, Mg²⁺ + Na⁺ + K⁺ + ATP, Mg²⁺ + K + ATP Mg²⁺. The number of high-affinity sites in the presence of Mg²⁺ + P_i 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 V_max values with the high and low concentrations of ATP were reduced 70% by phospholipase A treatment. The apparent K_m 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⁺ + P_i, 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⁺ + P_i, 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.

Note:
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.