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(+).

The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements

“…A rise in fluorescence was also observed as a function of choline chloride (ChCl), used to test ionic strength. In the case of the Na,K-ATPase, it has been established that certain protonated buffers favor the E1 conformation [19,[22][23][24]; thus, it is also conceivable for choline, to increase eosin fluorescence in the H,K-ATPase preparation. As Na + has been proposed as a substitute for H + in the H,K-ATPase [4,5], here we investigated its influence on the ATPase activity.…”

Caracterização cinética e molecular da (Na>sup<+>/sup<, K>sup<+>/sup<)-ATPase do tecido branquial de >i<Callinectes danae>/i< submetido a estresse por cobalto

“…O ortovanadato também inibide as P-ATPases na faixa nanomolar dada a sua estrutura tetraédrica ser altamente similar à do fosfato (Fedosova, Cornelius Klodos, 1998;Montes, Monti, Rossi, 2012). Esse inibidor se liga covalentemente ao resíduo de aspartato no sítio de fosforilação inibindo o ciclo catalítico, mantendo a enzima em um estado semelhante ao E2 (Fedosova, Cornelius, Klodos, 1998;Montes, Monti, Rossi, 2012), originando um complexo enzima-ortovanadato em uma conformação aberta ou fechada (Montes, Monti, Rossi, 2012).…”

Estudo bioquímico comparativo da (Na+ , K+)-ATPase branquial de Macrobrachium amazonicum (Heller, 1862) habitante de regiões continentais

“…Diagrama da ligação da ouabaína na subunidade  da (Na + , K + )-ATPase e estrutura química da ouabaína. Modificado de Aperia, 2012;Slingerland et al, 2013. O ortovanadato apresenta uma estrutura tetraédrica altamente semelhante à do fosfato e, devido a essa semelhança o ortovanadato inibe as P-ATPases (Boxenbaum et al, 1998;Fedosova et al, 1998;Montes et al, 2012). A inibição ocorre através da ligação do ortovanadato ao aspartato que é fosforilado no ciclo catalítico bloqueando o ciclo reacional e mantendo a enzima num estado semelhante a E2 (Fedosova et al, 1998;Montes et al, 2012).…”