info:eu-repo/semantics/article
Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC
Fecha
2011-01Registro en:
Montes, Monica Raquel; Spiaggi, Alejandro Javier; Monti, José Luis Eugenio; Cornelius, Flemming; Olesen, Claus; et al.; Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1808; 1; 1-2011; 316-322
0005-2736
CONICET Digital
CONICET
Autor
Montes, Monica Raquel
Spiaggi, Alejandro Javier
Monti, José Luis Eugenio
Cornelius, Flemming
Olesen, Claus
Garrahan, Patricio Jose
Rossi, Rolando Carlos
Resumen
Despite its similarity with the Na+/K+-ATPase, it has not been possible so far to isolate a K+-occluded state in the H+/K+-ATPase at room temperature. We report here results on the time course of formation of a state containing occluded Rb+ (as surrogate for K+) in H+/K+-ATPase from gastric vesicles at 25 °C. Alamethicin (a pore-forming peptide) showed to be a suitable agent to open vesicles, allowing a more efficient removal of Rb+ ions from the intravesicular medium than C12E8 (a non-ionic detergent). In the presence of vanadate and Mg2+, the time course of [86Rb]Rb+ uptake displayed a fast phase due to Rb+ occlusion. The specific inhibitor of the H+/K+-ATPase SCH28080 significantly reduces the amount of Rb+ occluded in the vanadate–H+/K+-ATPase complex. Occluded Rb+ varies with [Rb+] according to a hyperbolic function with K0.5 = 0.29 ± 0.06 mM. The complex between the Rb+-occluded state and vanadate proved to be very stable even after removal of free Mg2+ with EDTA. Our results yield a stoichiometry lower than one occluded Rb+ per phosphorylation site, which might be explained assuming that, unlike for the Na+/K+-ATPase, Mg2+-vanadate is unable to recruit all the Rb+-bound to the Rb+-occluded form of the Rb+–vanadate–H+/K+-ATPase complex.