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

SPIAGGI ALEJANDRO; ROLANDO ROSSI; MÓNICA R. MONTES

Salta

Congreso; 3rd Latin American Protein Society Meeting; 2010

Socieda latinoamerican de Proteínas

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 temperature1. 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 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 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 SCH280802 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 at 25 ºC yield a stoichiometry lower than one occluded Rb+ per phosphorylation site but this value increases at 4 ºC, 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.     References: 1. I.M. Glynn and S.J.D. Karlish, Annu. Rev. Biochem., 59  171-205 (1990). 2. J. Mendlein and G. Sachs, J. Biol. Chem., 265(9) 5030-5036 (1990).   With grants from ANPCyT, CONICET and University of Buenos Aires.