Molecular Pharmacology, Vol 10, 494-500, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Structure-Activity Relationships of Cardiotonic Steroids for the Inhibition of Sodium- and Potassium-Dependent Adenosine Triphosphatase

III. Dissociation Rate Constants of Various Enzyme—Cardiac Glycoside Complexes Formed in the Presence of Sodium, Magnesium, and Adenosine Triphosphate

¹ Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706

The dissociation rate constants (k_d) of cardiac monoglycoside—(Na⁺ + K⁺)-ATPase complexes formed in the Na⁺-Mg²⁺-ATP system (type I complex) were determined by enzymatic assay after dilution. The k_d value of each cardiac glycoside—enzyme complex thus formed was greater than that of the complex formed in the Mg²⁺-P_i system (type II complex). Whereas the k_d values of type II complexes were dependent only on the sugar moiety, the k_d values of type I complexes were affected by both the steroid and sugar moieties. For cardiac glycoside—enzyme complexes in which the sugar moiety was the same, the stability of type I complexes increased in the order: digitoxigenin glycoside < strophanthidin glycoside < digoxigenin glycoside and strophanthidin glycoside < ouabagenin glycoside. If the steroid moiety was the same, the stability increased in the order: digitoxide [unknown] 6-deoxyglucoside [unknown] fucoside < 6-deoxyguloside < rhamnoside. Methylation of the sugar 3'-hydroxyl group decreased stability. These data indicate that in type I complexes a sugarspecific site(s) on the enzyme binds the sugar moiety at the 2'-- and 3'- or -hydroxyl groups by hydrogen bond(s), and at the 5'--methyl group by a hydrophobic bond. The activation energy of this dissociation was approximately 30 kcal/mol with all the cardiac monoglycosides tested. The differences in k_d values between type I and type II complexes indicate that the two are distinct, possibly as a result of conformational differences in the sugar-specific site of the (Na⁺ + K⁺)-ATPase.

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ACKNOWLEDGMENT We thank Dr. Lowell E. Hokin for his interest and thoughtful help with the manuscript.