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
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 increas...
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| Autores principales: | , , |
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| Formato: | JOUR |
| Materias: | |
| Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_00052736_v1818_n9_p2087_Montes |
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| Sumario: | 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+. © 2012 Elsevier B.V. All rights reserved. |
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