RT Journal Article
SR Electronic
T1 Binding of Digoxigenin to Sodium- and Potassium-Dependent Adenosine Triphosphatase
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 399
OP 408
VO 12
IS 3
A1 ATSUNOBU YODA
YR 1976
UL http://molpharm.aspetjournals.org/content/12/3/399.abstract
AB [12α-3H]Digoxigenin was prepared by the reduction of 12-dehydrodigoxigenin with NaB3H4. Binding to the sodium- and potassium-dependent adenosine triphosphatase was studied at room temperature. Digoxigenin bound to the enzyme with high affinity. This binding was eliminated by prior ouabain treatment and was dependent on the presence of ligands. As in cardiac glycoside binding, mixtures of Mg2+ and Pi or of Na+, Mg2+, and ATP were both effective as ligands. Scatchard plots of binding were linear and showed that the dissociation constant did not change at different concentrations of each ligand except in the very low concentration range, but that the number of binding sites on the enzyme was reduced on descreasing the ligand concentration. The present results show that the binding of digoxigenin to the (Na+ + K+)-ATPase does not follow the usual equation representing a reversible reaction. At saturating concentrations of ligands, i.e., 2 mM Mg2+ and 2 mM Pi, or 50 mM Na+, 2 mM Mg2+, and 2 mM ATP, the number of binding sites was close to the number of ouabain binding sites for each set of ligands. The dissociation constant was 0.041 µM in the Mg2+-Pi system and 0.078 µM in the Na+-Mg2+-ATP system. These dissociation constants are lower than the I50 value of digoxigenin (0.4 µM) under the conditions used for assay. ACKNOWLEDGMENTS The author thanks Dr. Lowell E. Hokin for his kind help with the manuscript, Drs. W. W. Cleland and L. A. Fahien, for their valuable discussions, and Mrs. Mary Lochner, for preparation of beef microsomes.