绵羊Na⁺,K⁺-ATP酶α1同工型中的谷氨酸327是阳离子诱导构象变化的关键残基。

Glutamic acid 327 in the sheep alpha 1 isoform of Na+,K(+)-ATPase is a pivotal residue for cation-induced conformational changes.

作者信息

Johnson C L, Kuntzweiler T A, Lingrel J B, Johnson C G, Wallick E T

机构信息

Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, OH 45267-0575, USA.

出版信息

Biochem J. 1995 Jul 1;309 ( Pt 1)(Pt 1):187-94. doi: 10.1042/bj3090187.

DOI:10.1042/bj3090187

PMID:7619055

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1135818/

Abstract

The cation binding characteristics of the mutant E327A formed in the sheep alpha 1 isoform of the Na+,K(+)-ATPase were examined using [3H]ouabain binding as a function of monovalent cation concentrations. Equilibrium competition binding assays in the presence of Mg2+, inorganic phosphate and various amounts of unlabelled ouabain indicated that both wild-type sheep alpha 1 protein and the E327A mutant expressed in 3T3 cells had similar affinities for ouabain (KD = 1.53 and 1.31 nM respectively). Sodium inhibition of ouabain binding appeared competitive in both enzymes. However, binding of three Na+ ions was required to explain the steep character of the Na+ inhibition curve for the wild-type Na+,K(+)-ATPase (Ki = 12.8 +/- 1.6 mM), whereas the binding of two Na+ ions was detected for the mutant E327A (Ki = 19.2 +/- 2.5 mM). Potassium binding of [3H]ouabain binding displayed a partially competitive nature with Hill coefficients of 2 for both wild-type sheep alpha 1 (Ki = 0.743 +/- 0.044 mM) and E327A (Ki = 0.875 +/- 0.067 mM). At concentrations of K+ above 10 mM, the sheep alpha 1 competition curve levelled off whereas the inhibition curve for E327A displayed a stimulation in ouabain binding. This stimulation in [3H]ouabain binding also occurred with Rb+, Cs+ and Li+, but was never observed with choline or Na+, suggesting that this effect was not due to ionic strength. From these [3H]ouabain-binding studies, it is obvious that the mutant enzyme E327A in the presence of Mg2+, Pi and ouabain, interacts with monovalent cations in a unique fashion. One interpretation of these data is that the glutamic acid residue at position 327 is involved in a conformational transition induced by the binding of monovalent cations to the Na+,K+-ATPase and that this transition is inhibited by the mutation of E327A.

摘要

利用[³H]哇巴因结合作为单价阳离子浓度的函数，研究了在绵羊Na⁺,K⁺-ATP酶α1同工型中形成的突变体E327A的阳离子结合特性。在Mg²⁺、无机磷酸盐和各种量的未标记哇巴因存在下进行的平衡竞争结合试验表明，野生型绵羊α1蛋白和在3T3细胞中表达的E327A突变体对哇巴因具有相似的亲和力（KD分别为1.53和1.31 nM）。两种酶中钠对哇巴因结合的抑制均表现为竞争性抑制。然而，野生型Na⁺,K⁺-ATP酶的钠抑制曲线的陡峭特征需要结合三个Na⁺离子来解释（Ki = 12.8±1.6 mM），而突变体E327A检测到结合两个Na⁺离子（Ki = 19.2±2.5 mM）。[³H]哇巴因结合的钾结合表现出部分竞争性，野生型绵羊α1（Ki = 0.743±0.044 mM）和E327A（Ki = 0.875±0.067 mM）的希尔系数均为2。在K⁺浓度高于10 mM时，绵羊α1竞争曲线趋于平稳，而E327A的抑制曲线显示哇巴因结合增加。用Rb⁺、Cs⁺和Li⁺时[³H]哇巴因结合也出现这种增加，但胆碱或Na⁺从未观察到这种情况，表明这种效应不是由于离子强度。从这些[³H]哇巴因结合研究中可以明显看出，在Mg²⁺、Pi和哇巴因存在下，突变酶E327A以独特方式与单价阳离子相互作用。这些数据的一种解释是，327位的谷氨酸残基参与单价阳离子与Na⁺,K⁺-ATP酶结合诱导的构象转变，并且这种转变被E327A突变所抑制。