RESUMO
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 C(12)E(8) (a non-ionic detergent). In the presence of vanadate and Mg(2+), the time course of [(86)Rb]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 K(0.5)=0.29±0.06mM. The complex between the Rb(+)-occluded state and vanadate proved to be very stable even after removal of free Mg(2+) 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, Mg(2+)-vanadate is unable to recruit all the Rb(+)-bound to the Rb(+)-occluded form of the Rb(+)-vanadate-H(+)/K(+)-ATPase complex.
