RT Journal Article
SR Electronic
T1 The Interaction of Ouabagenin and (Na<sup>+</sup> + K<sup>+</sup>)-ATPase in the Presence of Na<sup>+</sup>, Mg<sup>2+</sup>, and ATP
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 62
OP 67
VO 19
IS 1
A1 ATSUNOBU YODA
A1 SHIZUKO YODA
YR 1981
UL http://molpharm.aspetjournals.org/content/19/1/62.abstract
AB (Na+ + K+)-ATPase-rich membrane fragments from the electric organ of the electric eel were used to study the association and dissociation of ouabagenin (a reversibly bound cardiac aglycone) in the Na+-Mg2+-ATP system. To measure these rates, one or three second assays for ATPase were performed using [γ-32P]ATP. This method assays the reversible binding, the binding of the steroid moiety. With this short-period assay, the pseudo-first order association rate constant (K'a) of 1 µM ouabagenin was found to be 0.78 min-1 at 30°—the same as that of 1 µM ouabain. However, the K'a of 1 µM ouabain was 0.40 min-1 by the dilution method which measures the pseudo-irreversible binding, the binding of the sugar moiety. These results support our previous hypothesis that the cardiac steroid binds with the enzyme by the steroid moiety at first, and the pseudo-irreversible binding of sugar moiety follows. In the dissociation of the ouabagenin-(Na+ + K+)-ATPase complex, the presence of K+ in the dissociation medium did not stabilize the complex, unlike the effect of K+ on the ouabain-enzyme complex. However, by elevating the pH value, the dissociation rate of the ouabagenin-enzyme complex was reduced, like that of the ouabain complex. Moreover, at pH 7.35, ouabagenin dissociates biphasically, and it is concluded that two forms of ouabagenin-(Na+ + K+)-ATPase complex exist in the Na+-Mg2+-ATP system, where the dissociation rate constants (Kd) of these complexes were 0.30 min-1 and 2.5 min-1 at 30°, respectively. Similar results were observed with the digoxigenin complex, where the Kd values were 0.46 min-1 and 3.1 min-1 at 30°.