Effect of magnesium ions on the high-affinity binding of eosin to the (Na+ + K+)-ATPase.

(1) The fluorescence of eosin Y in the presence of (Na+ + K+)-ATPase is enhanced by Mg2+. The enhancement by Mg2+ is larger than that obtained with Na+ (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232-240). Mg2+ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na+. (2) The Mg2+-dependent enhancement of fluorescence can be reversed by K+ as well as by ATP. In the presence of Mg2+ + Pi (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg2+ and a low concentration of K+ (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg2+ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K+-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per 32P-labelling site in the presence of 5 mM Mg2+ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 mM Mg2+ + 0.25 Pi, the Kd values are 0.14 microM and 1.3 microM, respectively. With 5 mM Mg2+, 150 mM Na+, the Kd values are 0.45 microM and 3.2 microM, respectively. With 5 mM Mg2+, the addition of K+ gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per 32P-labelling site). With 5 mM Mg2+ + 150 mM K+, the affinities of the two binding sites become identical, at a Kd of 17 microM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg2+-form to the K+-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na+ in the presence of Mg2+ does not appreciably change the rate of conversion to the K+-form, giving a rate constant of about 110 s-1. However, the addition of oligomycin in the presence of Mg2+ + Na+ had a profound effect: the rate of conversion to the K+-form was decreased by a factor of 2000 to about 0.063 s-1. This suggests that the conformation with Mg2+ alone is different from the conformation with Na+ alone. (6) The effects of K+, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per 32P-labelling site are bound to ATP sites.