Reactivity of cysteines in the transmembrane region of the Na, K-ATPase alpha subunit probed with Hg(2+).
Reactivity of cysteines in the transmembrane region of the Na, K-ATPase alpha subunit probed with Hg(2+).
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用 Hg(2 ) 探测 Na,K-ATPase α 亚基跨膜区半胱氨酸的反应性。
DOI：
10.1007/s002320010002
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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：
--
作者：
Zichittella,AE;Shi,HG;Argüello,JM
为了深入了解Na，K-ATP酶的结构和构象偶联，本研究利用小探针对α1亚基跨膜半胱氨酸的反应进行了表征。将表达异源Na，K-ATP酶的HeLa细胞置于磷酸化的E2P.Na/E2 P或去磷酸化的ATP. E1.K/ATP. E1两种构象中的任何一种，然后用（μm）HgCl 2处理。在这两种条件下，治疗导致酶失活后的双指数动力学哇巴因敏感的K+吸收测量确定。然而，当在有利于酶磷酸化的介质中探测蛋白质时，缓慢反应组分的速率常数大十倍。使用携带位于α1亚基跨膜区的半胱氨酸突变的酶来鉴定反应性SH基团。替换Cys 104 Ser通过在两种处理条件下去除慢反应组分来减少酶失活。置换Cys 964使Hg ~（2+）处理的去磷酸化酶的快反应组分失活速率常数降低79%，慢反应组分失活。此外，Cys 964 Ser取代酶在磷酸化条件下处理时对Hg 2+不敏感。这些结果表明，Cys 964参与了Hg 2+的快速失活。虽然双突变体Cys 964，104 Ser仍然是部分失活的治疗nonphosphorylating条件下，酶缺乏跨膜半胱氨酸是不敏感的Hg 2+在所有的治疗条件下。因此，这种酶提供了可以测试工程化跨膜半胱氨酸的可及性的背景。
To gain insight into the structure and conformational coupling in the Na,K-ATPase, this study characterized the reaction of the α1 subunit transmembrane cysteines with a small probe. Intact HeLa cells expressing heterologous Na,K-ATPase were treated with (μm) HgCl2after placing the enzyme predominantly in either of two conformations, phosphorylated E2P.Na/E2P or dephosphorylated ATP.E1.K/ATP.E1. Under both conditions the treatment led to enzyme inactivation following a double exponential kinetic as determined by ouabain-sensitive K+uptake measurements. However, the rate constant of the slow reacting component was ten times larger when the protein was probed in a medium that would favor enzyme phosphorylation. Enzymes carrying mutations of cysteines located in the α1 subunit transmembrane region were used to identify the reacting–SH groups. Replacement Cys104Ser reduced enzyme inactivation by removing the slow reacting component under both treatment conditions. Replacement of Cys964 reduced the inactivation rate constant of the fast reacting component (79%) and removed the slow reacting component when the dephosphorylated enzyme was treated with Hg2+. Moreover, Cys964Ser substituted enzyme was insensitive to Hg2+when treated under phosphorylation conditions. These results indicate that Cys964 is involved in the fast inactivation by Hg2+. Although the double mutant Cys964, 104Ser was still partially inactivated by treatment under nonphosphorylating conditions, an enzyme devoid of transmembrane cysteines was insensitive to Hg2+under all treatment conditions. Thus, this enzyme provides a background where accessibility of engineered transmembrane cysteines can be tested.