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Artículo

E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2

Montes, Monica RaquelIcon ; Monti, José Luis EugenioIcon ; Rossi, Rolando CarlosIcon
Fecha de publicación: 09/2012
Editorial: Elsevier Science
Revista: Biochimica et Biophysica Acta - Biomembranes
ISSN: 0005-2736
Idioma: Inglés
Tipo de recurso: Artículo publicado
Clasificación temática:
Biofísica

Resumen

This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb+ deocclusion stimulated by Na+ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb+ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb+ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb+]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb+-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na+-stimulated Rb+ deocclusion would require the release of at least one Rb+ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb+ in equilibrium conditions. Results show that, while Mg2 + decreases the affinity for Rb+, addition of vanadate offsets this effect, increasing the affinity for Rb+. In transient experiments, we investigated the exchange of Rb+ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na+ without significantly affecting the rate of Rb+ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb+ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb+.
Palabras clave: Na+/K+-Atpase , Conformational Transition , Rb+-Deocclusion , Vanadate
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info:eu-repo/semantics/openAccess Excepto donde se diga explícitamente, este item se publica bajo la siguiente descripción: Atribución-NoComercial-SinDerivadas 2.5 Argentina (CC BY-NC-ND 2.5 AR)
Identificadores
URI: http://hdl.handle.net/11336/18197
DOI: http://dx.doi.org/10.1016/j.bbamem.2012.04.001
Colecciones
Articulos(IQUIFIB)
Articulos de INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Citación
Montes, Monica Raquel; Monti, José Luis Eugenio; Rossi, Rolando Carlos; E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1818; 9; 9-2012; 2087-2093
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