在HeLa细胞中表达的大鼠Na，K - ATP酶α1、α2和α3亚型底物依赖性特性的比较。

Comparison of the substrate dependence properties of the rat Na,K-ATPase alpha 1, alpha 2, and alpha 3 isoforms expressed in HeLa cells.

作者信息

Jewell E A, Lingrel J B

机构信息

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.

出版信息

J Biol Chem. 1991 Sep 5;266(25):16925-30.

PMID:1653250

Abstract

The role of multiple isoforms for the alpha subunit of Na,K-ATPase is essentially unknown. To examine the functional properties of the three alpha subunit isoforms, we developed a system for the heterologous expression of Na,K-ATPase in which the enzymatic activity of each isoform can be independently analyzed. Ouabain-resistant forms of the rat alpha 2 and alpha 3 subunits were constructed by site-directed mutagenesis of amino acid residues at the extracellular borders of the first and second transmembrane domains (L111R and N122D for alpha 2 and Q108R and N119D for alpha 3). cDNAs encoding the rat alpha 1 subunit, which is naturally ouabain-resistant, and rat alpha 2 and alpha 3, which were mutated to ouabain resistance (designated rat alpha 2* and rat alpha 3*, respectively) were cloned into an expression vector and transfected into HeLa cells. Resistant clones were isolated and analyzed for ouabain-inhibitable ATPase activity in the presence of 1 microM ouabain, which inhibits the endogenous Na,K-ATPase present in HeLa cells (I50 approximately equal to 10 nM). The remaining activity corresponds to Na,K-ATPase molecules containing the transfected rat alpha 1, rat alpha 2*, or rat alpha 3* isoforms. Utilizing this system, we examined Na+, K+, and ATP dependence of enzyme activity. Na,K-ATPase molecules containing rat alpha 1 and rat alpha 2* exhibited a 2-3-fold higher apparent affinity for Na+ than those containing rat alpha 3* (apparent KNa+ (millimolar): rat alpha 1 = 1.15 +/- 0.13; rat alpha 2* = 1.05 +/- 0.11; rat alpha 3* = 3.08 +/- 0.06). Additionally, rat alpha 3* had a slightly higher apparent affinity for ATP (in the millimolar concentration range) compared with rat alpha 1 or rat alpha 2* (apparent K0.5 (millimolar): rat alpha 1 = 0.43 +/- 0.12; rat alpha 2* = 0.54 +/- 0.15; rat alpha 3* = 0.21 +/- 0.04) and all three isoforms has similar apparent affinities for K+ (apparent KK+: rat alpha 1 = 0.45 +/- 0.01; rat alpha 2* = 0.43 +/- 0.004; rat alpha 3* = 0.27 +/- 0.01). This study represents the first comparison of the functional properties of the three Na,K-ATPase alpha isoforms expressed in the same cell type.

摘要

钠钾-ATP酶α亚基多种同工型的作用基本上还不清楚。为了研究三种α亚基同工型的功能特性，我们开发了一种用于钠钾-ATP酶异源表达的系统，在该系统中每种同工型的酶活性都可以独立分析。通过对第一和第二跨膜结构域细胞外边界的氨基酸残基进行定点诱变，构建了大鼠α2和α3亚基的哇巴因抗性形式（α2的L111R和N122D以及α3的Q108R和N119D）。将编码天然对哇巴因抗性的大鼠α1亚基以及突变为对哇巴因抗性的大鼠α2和α3（分别命名为大鼠α2和大鼠α3）的cDNA克隆到表达载体中，并转染到HeLa细胞中。分离出抗性克隆，并在存在1微摩尔哇巴因的情况下分析其对哇巴因抑制的ATP酶活性，哇巴因可抑制HeLa细胞中存在的内源性钠钾-ATP酶（I50约等于10纳摩尔）。剩余的活性对应于含有转染的大鼠α1、大鼠α2或大鼠α3同工型的钠钾-ATP酶分子。利用该系统，我们研究了酶活性对Na+、K+和ATP的依赖性。含有大鼠α1和大鼠α2的钠钾-ATP酶分子对Na+的表观亲和力比含有大鼠α3的分子高2 - 3倍（表观KNa+（毫摩尔）：大鼠α1 = 1.15±0.13；大鼠α2* = 1.05±0.11；大鼠α3* = 3.08±0.06）。此外，与大鼠α1或大鼠α2相比，大鼠α3对ATP（在毫摩尔浓度范围内）的表观亲和力略高（表观K0.5（毫摩尔）：大鼠α1 = 0.43±0.12；大鼠α2* = 0.54±0.15；大鼠α3* = 0.21±0.04），并且所有三种同工型对K+的表观亲和力相似（表观KK+：大鼠α1 = 0.45±0.01；大鼠α2* = 0.43±0.004；大鼠α3* = 0.27±0.01）。这项研究首次比较了在同一细胞类型中表达的三种钠钾-ATP酶α同工型的功能特性。