Functional role of cysteine residues in the (Na,K)-ATPase alpha subunit.
Functional role of cysteine residues in the (Na,K)-ATPase alpha subunit.
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(Na,K)-ATPase α 亚基中半胱氨酸残基的功能作用。
DOI：
10.1016/s0005-2736(99)00245-x
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Argüello,JM
中科院分区：
文献类型：
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作者：
Shi,HG;Mikhaylova,L;Zichittella,AE;Argüello,JM
采用定点突变、表达和动力学分析方法研究了绵羊（Na，K）-ATP酶α1亚基中23个半胱氨酸的结构-功能作用。这些半胱氨酸中的二十个被丙氨酸或丝氨酸单独取代。Cys 452、Cys 455和Cys 456同时被丝氨酸取代。这些取代被引入到哇巴因抗性α1绵羊同种型中，并在哇巴因选择压力下在HeLa细胞中表达。用编码Cys 242替代物的cDNA转染的HeLa细胞在哇巴因选择压力下不能存活。单取代剩余的半胱氨酸产生功能酶，虽然有些降低营业额。作为这些替换的结果，在酶Na+和K+依赖性中仅观察到微小的变化。某些取代明显影响E1 Participe 2平衡，如ATP作用于其低亲和力结合位点的Km变化所示。这些结果表明，个别半胱氨酸，除了Cys 242，酶的功能是不必要的。此外，这表明假定的二硫键的存在不是α1亚基折叠和后续活性所必需的。构建了跨膜区缺少半胱氨酸残基的（Na，K）-ATP酶（Cys 104，138，336，802，911，930，964，983 Xxx）。结果表明，在该酶中，Na ~+或K ~+对（Na，K）-ATP酶的激活作用的K_（1/2）没有改变，但其周转率降低了50%。在高亲和力位点的ATP结合不受影响。然而，E1 Participe 2平衡向E1型的位移表明，在低亲和力位点的Km ATP伴随着增加的IC 50钒酸盐抑制的小幅下降。因此，跨膜半胱氨酸缺陷型（Na，K）-ATP酶在其与生理配体的相互作用中没有关键的改变。
The structural-functional roles of 23 cysteines present in the sheep (Na,K)-ATPase α1 subunit were studied using site directed mutagenesis, expression, and kinetics analysis. Twenty of these cysteines were individually substituted by alanine or serine. Cys452, Cys455 and Cys456 were simultaneously replaced by serine. These substitutions were introduced into an ouabain resistant α1 sheep isoform and expressed in HeLa cells under ouabain selective pressure. HeLa cells transfected with a cDNA encoding for replacements of Cys242 did not survive ouabain selective pressure. Single substitutions of the remaining cysteines yielded functional enzymes, although some had reduced turnover rates. Only minor variations were observed in the enzyme Na+and K+dependence as a result of these replacements. Some substitutions apparently affect the E1↔E2 equilibrium as suggested by changes in the Kmof ATP acting at its low affinity binding site. These results indicate that individual cysteines, with the exception of Cys242, are not essential for enzyme function. Furthermore, this suggests that the presence of putative disulfide bridges is not required for α1 subunit folding and subsequent activity. A (Na,K)-ATPase lacking cysteine residues in the transmembrane region was constructed (Cys104, 138, 336, 802, 911, 930, 964, 983Xxx). No alteration in the K1/2of Na+or K+for (Na,K)-ATPase activation was observed in the resulting enzyme, although it showed a 50% reduction in turnover rate. ATP binding at the high affinity site was not affected. However, a displacement in the E1↔E2 equilibrium toward the E1 form was indicated by a small decrease in the Kmof ATP at the low affinity site accompanied by an increase in IC50for vanadate inhibition. Thus, the transmembrane cysteine-deficient (Na,K)-ATPase appears functional with no critical alteration in its interactions with physiological ligands.