Segall Laura, Javaid Zahid Z, Carl Stephanie L, Lane Lois K, Blostein Rhoda
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1A4, Canada.
J Biol Chem. 2003 Mar 14;278(11):9027-34. doi: 10.1074/jbc.M211636200. Epub 2003 Jan 14.
We showed earlier that the kinetic behavior of the alpha2 isoform of the Na,K-ATPase differs from the ubiquitous alpha1 isoform primarily by a shift in the steady-state E(1)/E(2) equilibrium of alpha2 in favor of E(1) form(s). The aim of the present study was to identify regions of the alpha chain that confer the alpha1/alpha2 distinct behavior using a mutagenesis and chimera approach. Criteria to assess shifts in conformational equilibrium included (i) K(+) sensitivity of Na-ATPase measured at micromolar ATP, under which condition E(2)(K(+)) --> E(1) + K(+) becomes rate-limiting, (ii) changes in K'(ATP) for low affinity ATP binding, (iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate of the partial reaction E(1)P --> E(2)P. We first confirmed that interactions between the cytoplasmic domains of alpha2 that modulate conformational shifts are fundamentally similar to those of alpha1, suggesting that the predilection of alpha2 for E(1) state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the alpha1/alpha2 chimeras indicates that the difference in E(1)/E(2) poise of the two isoforms cannot be accounted for by their notably distinct N termini, but rather by the front segment extending from the cytoplasmic N terminus to the C-terminal end of the extracellular loop between transmembranes 3 and 4, with a lesser contribution of the alpha1/alpha2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E(1) shift of alpha2 results primarily from differences in the conformational transition of the dephosphoenzyme, (E(2)(K(+)) --> E(1) + K(+)), rather than phosphoenzyme (E(1)P --> E(2)P).
我们之前表明，钠钾ATP酶α2亚型的动力学行为与普遍存在的α1亚型不同，主要在于α2的稳态E(1)/E(2)平衡发生了偏移，有利于E(1)形式。本研究的目的是使用诱变和嵌合体方法来确定α链中赋予α1/α2不同行为的区域。评估构象平衡变化的标准包括：(i) 在微摩尔ATP浓度下测量的钠ATP酶对K(+)的敏感性，在此条件下E(2)(K(+))→E(1)+K(+)成为限速步骤；(ii) 低亲和力ATP结合的K'(ATP)变化；(iii) 钠钾ATP酶活性对钒酸盐的敏感性；以及(iv) 部分反应E(1)P→E(2)P的速率。我们首先证实，调节构象变化的α2胞质结构域之间的相互作用与α1的基本相似，这表明α2对E(1)状态的偏好是由于两种亚型一级结构的差异。α1/α2嵌合体的动力学行为表明，两种亚型E(1)/E(2)平衡的差异不能由它们明显不同的N末端来解释，而是由从胞质N末端延伸到跨膜3和4之间细胞外环C末端的前端片段来解释，ATP结合结构域附近M4-M5环内α1/α2不同部分的贡献较小。此外，我们表明α2的E(1)偏移主要源于去磷酸化酶构象转变（E(2)(K(+))→E(1)+K(+)）的差异，而非磷酸化酶（E(1)P→E(2)P）。
