Mammalian alpha 1-subunit of Na(+)-K(+)-ATPase does not need its amino terminus to maintain cell viability.
Mammalian alpha 1-subunit of Na(+)-K(+)-ATPase does not need its amino terminus to maintain cell viability.
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哺乳动物 Na()-K()-ATP 酶的 α1 亚基不需要其氨基末端来维持细胞活力。
DOI：
10.1152/ajpcell.1994.267.2.c590
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Pressley,TA
中科院分区：
文献类型：
--
作者：
Shanbaky,NM;Pressley,TA
Na(+)-K(+)-腺苷三磷酸酶 (ATPase) 的催化 α 亚基的氨基末端在迄今为止测序的各种亚型和物种中并不保守，但它始终包含富含赖氨酸的基序。为了研究这个高电荷区域所发挥的功能作用，我们改变了大鼠α1的氨基末端并评估了突变体维持细胞活力的能力。编码来自人c-myc癌基因产物的10个氨基酸表位的核苷酸序列被替换为编码α1的前31个氨基酸的野生型序列。然后将含有myc替换的嵌合cDNA引入哇巴因敏感的猴肾细胞中。哇巴因的选择产生了可行的菌落，这表明引入的突变体是有功能的，并赋予大鼠哇巴因抗性表型，尽管从其氨基末端去除了高电荷区域。随后的酶分析证实了受体菌落中存在哇巴因的低亲和力结合位点，免疫印迹揭示了膜部分中表达的多肽上的 myc 表位。这些结果表明，前 31 个氨基酸不是 α1 功能所必需的，并且与氨基末端相关的翻译后裂解是不必要的。
The amino terminus of the catalytic alpha-subunit from Na(+)-K(+)-adenosinetriphosphatase (ATPase) is not conserved among the various isoforms and species sequenced to date, yet it always includes a lysine-rich motif. To investigate the functional role played by this highly charged region, we altered the amino terminus of rat alpha 1 and evaluated the ability of the mutant to sustain cell viability. Nucleotide sequence encoding a 10-amino acid epitope from the human c-myc-oncogene product was substituted for the wild-type sequence encoding the first 31 amino acids of alpha 1. The chimeric cDNA containing the myc substitution was then introduced into ouabain-sensitive monkey kidney cells. Selection in ouabain produced viable colonies, suggesting that the introduced mutant was functional and conferred the ouabain-resistant phenotype of rats, despite the removal of the highly charged region from its amino terminus. Subsequent enzymatic analysis confirmed the presence of low-affinity binding sites for ouabain in the recipient colonies, and immunoblotting revealed the myc epitope on the expressed polypeptides in a membrane fraction. These results suggest that the first 31 amino acids are not required for function of alpha 1 and that the posttranslational cleavage associated with the amino terminus is unnecessary.