Ouabain binding to phospholipid-dependent adenosine triphosphatase.
The role of phospholipid in the binding of ouabain to the (Na+ + K+)-dependent adenosine triphosphatase was studied. Enzyme preparations obtained from rabbit kidney were treated with Lubrol WX to remove the phospholipid component essential for ATPase activity. Reconstituted enzyme samples were prepared by the addition of phosphatidylserine and sedimentation of an enzymically active lipid-protein complex. The binding of ouabain to both kinds of preparations was measured under equilibrium conditions with the use of 3H-labelled ouabain and initial ouabain concentrations in the range 0.01-1 micrometer. The main findings were: (i) (Mg2+ + Pi) promoted binding of significant quantities of ouabain only to the reconstituted enzyme; (ii) the absence of added Na+, (Mg2+ + ATP) similarly promoted binding only to the reconstituted samples; (iii) the addition of Na+ in the presence of (Mg2+ + ATP) increased the amount of ouabain bound to the reconstituted enzyme when the ouabain concentration was below about 0.1 micrometer, but it had no effect when the ouabain concentration was about 1 micrometer; (iv) (Mg2+ + ATP) induced ouabain binding to the depleted enzyme only when Na+ was also added; (v) the amount of ouabain bound to both depleted and reconstituted enzymes was the same in the presence of (Mg2+ + ATP + Na+); (vi) the reconstituted enzyme appeared to have a greater affinity for Na+ than did the depleted enzyme.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D010042:Ouabain / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism
D008024:Ligands; D010710:Phosphates; D010743:Phospholipids; D010042:Ouabain; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D012964:Sodium; D000251:Adenosine Triphosphatases; D008274:Magnesium; D011188:Potassium