Daly S E, Lane L K, Blostein R
Department of Medicine, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 1994 Sep 30;269(39):23944-8.
One region of marked sequence diversity among the highly homologous alpha isoforms of the Na,K-ATPase is the lysine-rich NH2 terminus. Expression of a mutant cDNA encoding an alpha 1 protein, minus the 32 NH2-terminal residues, results in a modified enzyme (alpha 1M32), which behaves similarly to alpha 1 in overall Na/K exchange activity (Vmax) and apparent affinities for intracellular Na+ and extracellular K+. However, with membranes isolated from HeLa cells expressing the rat alpha 1M32 mutant, as well as membranes from cells expressing the rat alpha 1 and the ouabain-resistant mutated forms of rat alpha 2 (alpha 2*) and alpha 3 (alpha 3*) developed by Jewell and Lingrel (Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930), distinct Na,K-ATPase kinetics are observed. Thus, at 1 microM ATP, the effects of K+ on the Na-ATPase activity of alpha 2* and alpha 1M32 are similar; both are activated, whereas alpha 1 and alpha 3 are inhibited by the addition of K+ at low (0.1 mM) concentration. These effects are attributed to different rates of a step involved in K+ deocclusion (E2(K)<-->E1K<-->E1 + K+) and are consistent with our earlier evidence (Wierzbicki, W., and Blostein, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 70-74) for a role of the NH2 terminus in the K+ deocclusion pathway of the Na,K-ATPase reaction. These differences are not directly related to differences in apparent affinities for ATP, since alpha 3* has alpha 1-like high affinity K+ inhibition but resembles alpha 2* and alpha 1M32 with respect to a lower K'ATP. Na-ATPase activities of alpha 2*, alpha 3*, and alpha 1M32, but not alpha 1, are activated by Li+ but not Rb+, consistent with a relatively faster rate of Li+ deocclusion (Post, R. L., Hegyvary, C., and Kume, S. (1972) J. Biol. Chem. 247, 6530-6540), as well as higher affinity of alpha 3 for extracellular K+ (Li+) activation of dephosphorylation (E2P + K+<-->E2(K) + Pi). Inhibition of Na-ATPase by higher concentrations (> or = 1 mM) K+ is observed with all isoforms and is attributed to K+ acting at inhibitory cytoplasmic sites.(ABSTRACT TRUNCATED AT 400 WORDS)
钠钾-ATP酶高度同源的α同工型中，一个具有显著序列多样性的区域是富含赖氨酸的氨基末端。编码α1蛋白（去除32个氨基末端残基）的突变cDNA的表达，产生了一种修饰酶（α1M32），其在整体钠/钾交换活性（Vmax）以及对细胞内Na⁺和细胞外K⁺的表观亲和力方面，表现与α1相似。然而，在用表达大鼠α1M32突变体的HeLa细胞膜，以及用表达大鼠α1和由朱厄尔和林格雷尔构建的大鼠α2（α2*）和α3（α3*）的哇巴因抗性突变形式的细胞膜（朱厄尔，E. A.，和林格雷尔，J. B.（1991年）《生物化学杂志》266，16925 - 16930）时，观察到了不同的钠钾-ATP酶动力学。因此，在1微摩尔ATP时，K⁺对α2和α1M32的钠-ATP酶活性的影响相似；二者均被激活，而在低浓度（0.1毫摩尔）下添加K⁺时，α1和α3被抑制。这些影响归因于K⁺去封闭过程（E2(K)⇌E1K⇌E1 + K⁺）中一个步骤的不同速率，并且与我们早期的证据（维尔兹比茨基，W.，和布洛斯坦，R.（1993年）《美国国家科学院院刊》90，70 - 74）一致，即氨基末端在钠钾-ATP酶反应的K⁺去封闭途径中起作用。这些差异与对ATP的表观亲和力差异没有直接关系，因为α3具有类似α1的高亲和力K⁺抑制作用，但在较低的K'ATP方面类似于α2和α1M32。α2、α3*和α1M32（而非α1）的钠-ATP酶活性被Li⁺激活但不被Rb⁺激活，这与Li⁺相对较快的去封闭速率一致（波斯特，R. L.，赫吉瓦里，C.，和久米，S.（1972年）《生物化学杂志》
