Effects of ATP and protons on the Na : K selectivity of the [formula omitted] studied by ligand effects on intrinsic and extrinsic fluorescence

Abstract

The effect of pH and of ATP on the Na : K selectivity of the ( Na + + K +)- ATPase has been tested under equilibrium conditions. The Na + : K +-induced change in intrinsic tryptophan fluorescence and in fluorescence of eosin maleimide bound to the system has been used as a tool. 1 mol of eosin maleimide per mol of enzyme gives no loss in either ATPase or phosphatase activity and the fluorescence in the presence of Na + is about 30% higher than in the presence of K +. Choline, protonated Tris, protonated histidine and Mg 2+ have an ‘Na +’ effect on the extrinsic fluorescence, while Rb +, Cs + and NH 4 + have a ‘K +’ effect. Choline and protonated Tris have an Na + effect on intrinsic fluorescence. A close correlation between the effect of Na + compared to K + on the fluorescence change and on Na + activation of hydrolysis indicates that the observed changes in fluorescence are due to an effect of Na + and of K + on the internal sites of the system. The equilibrium between the two conformations, which are reflected by the difference in fluorescence with Na + and K +, respectively, is highly influenced by the concentration of protons. At a given Na + : K + ratio, an increase in the proton concentration shifts the equilibrium towards the ‘K +’ fluorescence form while a decrease shifts the equilibrium towards the ‘Na +’ fluorescence form, i.e., protons increase the apparent affinity for K + and vice versa, K + increases pK values of importance for the Na + : K + selectivity. Conversely, a decrease in protons increases the apparent affinity for Na + and vice versa, Na + decreases the pK . ATP decreases the apparent pK for the protonation-deprotonation, i.e., ATP facilitates the deprotonation which accompanies Na + binding. The results suggest two effects of ATP for the hydrolysis in the presence of Na + and K +: (i) at low ATP concentrations ( K 0.5 < 10 μ M ) on the K +-Na + exchange on the internal sites and (ii) at higher, substrate, concentrations on the activation by K + on the external sites.