title: E2→E1 transition and Rb(+) release induced by Na(+) in the Na(+)/K(+)-ATPase. Vanadate as a tool to investigate the interaction between Rb(+) and E2.
abstract: This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb(+) deocclusion stimulated by Na(+) in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb(+) and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb(+) deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb(+)]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb(+)-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na(+)-stimulated Rb(+) deocclusion would require the release of at least one Rb(+) ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb(+) in equilibrium conditions. Results show that, while Mg(2+) decreases the affinity for Rb(+), addition of vanadate offsets this effect, increasing the affinity for Rb(+). In transient experiments, we investigated the exchange of Rb(+) between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na(+) without significantly affecting the rate of Rb(+) deocclusion. On the other hand, we found the first evidence of a very low rate of Rb(+) occlusion in the enzyme-vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb(+).
authors.affiliation: Departamento de Quimica Biologica, Universidad de Buenos Aires, Buenos Aires, Argentina. mmontes@qb.ffyb.uba.ar
authors.forename: Mónica R
authors.initials: MR
authors.lastname: Montes
authors.forename: José L E
authors.initials: JL
authors.lastname: Monti
authors.forename: Rolando C
authors.initials: RC
authors.lastname: Rossi
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001703:Biophysics / Q000379:methods; D004801:Eosine Yellowish-(YS) / Q000737:chemistry; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008954:Models, Biological; D011485:Protein Binding; D011487:Protein Conformation; D012413:Rubidium / Q000737:chemistry / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013552:Swine; D013997:Time Factors; D014638:Vanadates / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D014638:Vanadates; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D012413:Rubidium; D004801:Eosine Yellowish-(YS)