INVESTIGADORES
GONZALEZ LEBRERO Rodolfo Martin
artículos
Título:
A parallel study of eosin-fluorescence change and Rb+ occlusion in the Na+/K+-ATPase.
Autor/es:
MÓNICA R, MONTES; RODOLFO MARTIN GONZALEZ LEBRERO; PATRICIO J, GARRAHAN; ROLANDO CARLOS, ROSSI
Revista:
ANNALS OF THE NEW YORK ACADEMY OF SCIENCES.
Editorial:
BLACKWELL PUBLISHING
Referencias:
Lugar: Oxford; Año: 2003 vol. 986 p. 298 - 300
ISSN:
0077-8923
Resumen:
The aim of this work is to study the correlation between the E1  E2 conformational transition and K+ occlusion. Measurement of the conformational transition was performed using the fluorescent probe Eosin Y (Skou and Esmann, 1981, Biochim. Biphys Acta, 647, 232). To measure occlusion we used the K+ congener Rb+. As eosin binds non-covalently to the enzyme, we explored the interactions between the probe and Rb+ with the Na+/K+-ATPase, both in equilibrium and transient conditions. Results from equilibrium experiments showed that at constant [Rb+], eosin decreases the amount of occluded Rb+ to a value that depends on [Rb+]. A maximum of two molecules of eosin are able to bind both to the free enzyme and to enzyme containing Rb+. Transient kinetic experiments showed that eosin decreases the rate of formation of occluded Rb+ and increases the rate of Rb+ release. The latter effect occurs as if eosin acted as an ATP analog, accelerating the transition E2  E1 but less effectively than the nucleotide, since addition of 1 mM Eosin inhibits the acceleration produced by 100 µM ATP on Rb+ release. In parallel experiments we looked at the time courses of change of eosin fluorescence and of occlusion of 86Rb+.produced by addition of Rb+ in media with 0.4 µM eosin and different concentrations of Rb+. The change in fluorescence and the formation of occluded Rb+ followed bi-exponential functions of time, and the corresponding parameters of their fitted curves were very similar, for all [Rb+]. These results agree with the hypothesis that Rb+ occlusion occurs concomitantly with the E1  E2 conformational transition.With grants from ANPCyT, UBA and CONICET, Argentina.