-“Tight binding of Rb+ in H+/K+-ATPase-enriched membrane vesicles”

MÓNICA R. MONTES, ALEJANDRO J. SPIAGGI, FLEMING CORNELIUS, PATRICIO J. GARRAHAN AND ROLANDO C. ROSSI.

Aarhus, Dinamarca

Congreso; 12th International Conference on Na,K-ATPase and Related Cation Pumps; 2008

International Conference on Na,K-ATPase and Related Cation Pumps

In this work we report studies on the binding and transport of Rb+ (acting as a K+ congener) in gastric vesicles from pig stomach (1) enriched in H+/K+-ATPase. It is generally believed that the reaction cycle of the different P-type ATPases involves the formation of intermediates in which the transported ions are held in an occluded state. The studies reported here are an attempt to determine if the H+/K+-ATPase is able to occlude Rb+ and if so to characterize the intermediate holding the occluded cation. To do this, we used a method, developed in our lab (2), that allowed us to isolate in a millisecond time scale species containing tightly bound cation.The ability of Rb+ to act as a K+ congener was assessed by measuring the fluorescence of eosin-bound enzyme. We found that, as it happens with K+, the fluorescence signal decreased in the presence of Rb+, apparently because it drives the enzyme into the E2 state. Likewise, Na+ seems to act as a congener of H+ since it caused an increase of the fluorescence level. In the experiments carried out to study transport and binding in pre-steady state, the enzyme preparation (0.05mg/ml) was incubated in media with [86Rb]RbCl, in the absence of ATP, during different times. We found that the time course of Rb+ uptake by the vesicles could be fitted by two exponential functions of time. The faster of these functions could in part represent Rb+ occlusion while the other exponential could describe Rb+ influx into the vesicles. In samples incubated with C12E8 to increase the fraction of unsealed vesicles, the presence of vanadate enhanced the amplitude of the fast component of Rb+ uptake but decreased the slow one. This effect might be the consequence of an increase in the amount of the intermediate holding occluded Rb+ and a decrease in the entry of the cation into the vesicles. This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas and Universidad de Buenos Aires, Argentina.  1- F. Cornelius and Y.A. Mahmmoud (2003) Biophys. J.:84:1690-1700 2- Rossi, R.C., Kaufman, S.B., González-Lebrero, R.M., Nørby, J.G., and Garrahan, P.J. (1999) Anal. Biochem. 270:276-285. 3- Klodos I. (2003) Ann.N.Y.Acad-Sci. 986:306-307