A quantitative analysis of the interaction between the fluorescent probe eosin and the Na+/K+ ATPase studied through Rb+ occlusion

MÓNICA R. MONTES, RODOLFO M. GONZÁLEZ-LEBRERO, PATRICIO J. GARRAHAN AND ROLANDO C. ROSSI

BIOCHEMISTRY

American Chemical Society

Año: 2004 vol. 43 p. 2062 - 2069

0006-2960

We report a study on the effect of the fluorescent probe eosin on some of the reactions involved in the conformational transitions that lead to the occlusion of the K+-congener Rb+ in the Na+/K+-ATPase. Eosin decreases the equilibrium levels of occluded Rb+, being this effect fully attributable to a decrease in the apparent affinity of the enzyme for Rb+ since the capacity for occlusion remains independent of eosin concentration. The results can be quantitatively described by a model that assumes that two molecules of eosin are able to bind to the Na+/K+-ATPase, both to the Rb+-free and to the Rb+-occluded enzyme regardless of the degree of cation occlusion. Concerning the effect on the affinity for Rb+ occlusion, transient state experiments show that eosin reduces the initial velocity of occlusion, and that like ATP, it increases the velocity of deocclusion of Rb+. Interactions between eosin and ATP on Rb+-release experiments seem to indicate that eosin binds to the low affinity site of ATP from which it exerts effects that are similar to those of the nucleotide.