Rb+ occlusion in the H+/K+-ATPase from gastric vesicles

SPIAGGI AJ; ROSSI RC; MONTES MR

Salta

Congreso; SAB 2010-Workshop CeBEM-Protein Society Meeting; 2010

Despite its similarity with the Na+/K+ ATPase, it has not been possible so far toisolate 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 forK+) in H+/K+ ATPase from gastric vesicles at 25 ºC. 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 presenceof vanadate and Mg2+, the time course of [86Rb]Rb+ uptake displayed a fast phase dueto Rb+ occlusion. The specific inhibitor of the H+/K+ ATPase SCH28080 significantlyreduces the amount of Rb+ occluded in the vanadate- H+/K+ ATPase complex. OccludedRb+ 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 evenafter removal of free Mg2+ with EDTA. Our results at 25 ºC yield a stoichiometry lowerthan one occluded Rb+ per phosphorylation site but this value increases at 4 ºC, whichmight be explained assuming that, unlike for the Na+/K+ ATPase, Mg2+-vanadate is unableto recruit all the Rb+-bound to the Rb+-occluded form of the Rb+-vanadate- H+/K+ ATPasecomplex.