Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC

MONTES MR; SPIAGGI AJ; MONTI, JLE; CORNELIUS F; OLESEN C; GARRAHAN PJ; ROSSI, RC

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Año: 2010 vol. 1808 p. 316 - 322

0005-2736

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 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 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. <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} -->