(Na+, K+)‐Activated Adenosinetriphosphatase of Axonal Membranes, Cooperativity and Control: Steady‐State Analysis

Abstract

The ATP sites. Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations. The (Na+, K+)‐activated ATPase displays Michaelis‐Menten kinetics for ATP under standard concentratio1 conditions of Na+ (100 mM) and K+ (10 mM). The steady‐state kinetics behavior changes at very low concentrations of K+ where negative co‐operativity is observed. The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p‐nitrophenylphosphate in the presence of Na+ and K+. The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 7.5. The Na+ sites. The binding of Na+ to its specific stimulatory sites (internal sites) is charaterized by positive cooperativity with a Hill coefficient nH(Na+) = 2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. The K+ sites. (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)‐ATPase activity as well as the p‐nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP). Binding is characterized by a Hill coefficient of 1.0 and a K 0.5(K+)= 0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. (b) The analysis of the p‐nitrophenylphosphatase or of the 2,4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions. The characteristics of stimulation in that case are, K0.5= 5 mM, nH= 1.9. The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site. Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites. Thirdly, this family of sites disappears in the presence of ATP, p‐nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites. Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+. Mg2+ stimulation of the (Na+, K+)‐ATPase is characterized by a Hill coefficient nH(Mg2+)= 1.0 and a K0.5(Mg2+)= 1 mM stimulation is essentially a V effect. Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small. Heterotropic interactions between the Mg2+, Na+ and K+ sites are also small. The fluidity of membrane lipids also controls the (Na+, K+)‐ATPase activity. Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane. Only the rate of the catalytic transformation is affected. Copyright © 1976, Wiley Blackwell. All rights reserved