Recent advances in cardiac glycoside-Na+,K+-ATPase interaction

Na+,K+-ATPase has been purified from lamb kidney and consists of two polypeptide peaks on polyacrylamide gel electrophoresis with an enzyme activity of 1,000 mumole Pi/mg pro per hr. A scheme depicting the interaction of cardiac glycoside with the enzyme and ligand effects on binding has been constructed. Under all ligand conditions, ouabain binding tends to reach the same maximum if sufficient ouabain is present. Initial rates vary with ligand conditions. Using a chase method, the rate of dissociation of the glycoside from the enzyme is not influenced by the ligands present, although with separation of the enzyme-glycoside complex from the binding medium, differences are noted. The effect of ouabain on Na binding demonstrated two classes of sites, KD = 0.2 mM and KD = 18 mM. Denaturation decreased the high affinity sites. There was also a good correlation between ouabain binding and inhibition of Na binding. Clearly, ligands are critical in regulating cardiac glycoside interaction with the enzyme.