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

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Abstract
[Axonal plasma membranes from walking leg nerves of Cancer paqurus were used.] Homotropic interactions between ATP sites were studied in a very large range of Na+ and K+ concentrations. The (Na+,K+)-activated ATPase [EC 3.6.1.3] displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM). The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed. The existence of 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 binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient nH(Na+) = 2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. Binding of K+ to the (external) stimulatory site of the ATPase was 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 K0.5(K+) = 0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. 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: 1st, saturation of the low affinity sites(s) by K+ prevents ATP binding to its high affinity internal site; 2nd, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites; 3rd, 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.