Abstract

The dissociation rate constants (k_d) of (Na⁺ + K⁺)-ATPase (EC 3.6.1.3) complexes with digitoxigenin or digoxigenin oligodigitoxides were determined by enzymatic assay after dilution. The k_d value of each cardiac glycoside—enzyme complex formed in the Na⁺-Mg²⁺-ATP system (type I complex) was greater than that of the complex formed in the Mg²⁺-P_[unknown] system (type II complex). In type I complexes the k_d value of the digitoxigenin oligodigitoxide complex was greater than that of the digoxigenin oligodigitoxide complex, but this difference was diminished in the type II complex. For a given aglycone and type of complex, the k_d values decreased in the order monodigitoxide > bisdigitoxide > tridigitoxide [unknown] tetradigitoxide. The activation energies of these dissociation constants were approximately 30 kcal/mole for the type I complex and 25 kcal/mole for the type II complex and did not change with the number of sugar moieties or the nature of the aglycone. The k_d values of the type II complexes were increased with Na⁺ plus ATP in the dilution medium in a manner similar to those of the monoglycosides. On the other hand, K⁺ in the dilution medium reduced the k_d values of only the type I complexes containing cardiac oligodigitoxides. These k_d values fell below those of the type II complex, in contrast to the situation with the cardiac monoglycosides. These results suggest that the second and third digitoxose moieties of the cardiac digodigitoxides can bind to the enzyme but that binding by the first digitoxose moiety is predominant.