The Effect of Ionic Strength and Specific Anions on Substrate Binding and Hydrolytic Activities of Na,K-ATPase

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP(3−), eosin(2−), p-nitrophenylphosphate, and V(max) for Na,K-ATPase and K(+)-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0–0.5 M of NaCl, Na(2)SO(4), and NaNO(3) and in 0.1 M Na-acetate, NaSCN, and NaClO(4). The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl ≈ Na(2)SO(4) ≈ Na-acetate < NaNO(3) < NaSCN < NaClO(4). We treated the influence of NaCl and Na(2)SO(4) on K (diss) for E·ADP as a “pure” ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO(3) (−) effect was compatible with competitive binding of NO(3) (−) and ADP in addition to the general I-effect. K (diss) for E·NO(3) was ∼32 mM. Analysis of V(max)/K (m) for Na,K-ATPase and K(+)-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.