E2鈫?span style='font-style: italic'>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

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• 作者：M&oacute ; nica R. Montes ; ^{mmontes@qb.ffyb.uba.ar} ; Jos&eacute ; L.E. Monti¹ ; Rolando C. Rossi
• 关键词：Na+/K+-ATPase ; Conformational transition ; Rb+-deocclusion ; Vanadate
• 刊名：Biochimica et Biophysica Acta (BBA)/Biomembranes
• 出版年：2012
• 期刊代码：14_00052736
• 类别：bio
• 出版时间：September, 2012
• 卷：1818
• 期：9
• 页码：2087-2093
• 文件大小：998 K

摘要

This work presents a detailed kinetic study that shows the coupling between the <em>Eem>2鈫?em>Eem>1 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 <em>Eem>1) as a function of time. The <em>Eem>2鈫?em>Eem>1 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 <em>Eem>2鈫?em>Eem>1 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 <em>Eem>2鈫?em>Eem>1 transition. Using vanadate to stabilize <em>Eem>2, 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 <em>Eem>2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the <em>Eem>2鈫?em>Eem>1 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⁺.