The nature of the transport ATPase—Digitalis complex: III. Rapid binding studies and effects of ligands on the formation and stability of magnesium plus phosphate-induced glycoside—Enzyme complex

Abstract

The [ 3H]ouabain-Na +,K +-ATPase complex formed in the presence of magnesium and inorganic phosphate (Complex II) may represent a different conformation than that formed in the presence of ATP, magnesium, and sodium (Complex I). The nomenclature, Complex I and II, are operational in nature. The differences in binding may, e.g., be due to different levels of phosphorylation and/or to different conformations of the enzyme prior to binding. The kinetics of binding under conditions that lead to Complex II formation can be characterized by a hyperbolic curve arbitrarily divided into three phases. Sodium and potassium exert differential effects on these three phases of binding, with sodium having a greater inhibitory effect than potassium. Potassium primarily retards the reaction. When added to the binding medium (Complex II) after maximal binding, sodium and potassium are found not only to dissociate a portion of the glycoside-enzyme complex, but also appear to convert the unbound enzyme into a conformation which may bind only a small amount of glycoside.