Van Huysse J W, Jewell E A, Lingrel J B
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
Biochemistry. 1993 Jan 26;32(3):819-26. doi: 10.1021/bi00054a012.
Chemical modification and proteolytic digestion studies have identified a transmembrane glutamic acid residue (E953) of the alpha subunit of the pig kidney Na, K-ATPase as a possible cation binding site [Goldshleger et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6911-6915]. In addition, an adjacent glutamate (E954) is conserved in all species and isoforms and may also be involved in cation binding. To further explore the role of these residues in ion transport, we have utilized a mutagenesis-expression strategy. This approach avoids the introduction of a large chemical moiety into the protein and allows specific amino acid substitutions to be introduced. Glutamic acid residues 955 and 956 of the rat alpha-1 subunit (corresponding to glutamates 953 and 954 of the pig kidney Na, K-ATPase) were replaced separately and together using site-directed mutagenesis of the rat alpha-1 cDNA. The mutant cDNAs were expressed in ouabain-sensitive HeLa cells. This system makes it possible to rapidly identify amino acid substitutions which significantly impair enzyme function, as substitutions which do not affect enzyme activity will yield colonies in the presence of ouabain, while substitutions which severely impair function will prevent or limit growth of the ouabain-sensitive HeLa cells. The amino acid replacements (E955Q, E956Q, E955Q-E956Q, E955D-E956D) all resulted in the growth of ouabain-sensitive cells, demonstrating that the modified Na, K-ATPase in each case was functional. To further study the altered enzymes, ouabain-resistant colonies were isolated and expanded into stable cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)
化学修饰和蛋白酶消化研究已确定猪肾钠钾ATP酶α亚基的一个跨膜谷氨酸残基（E953）为可能的阳离子结合位点[戈尔德施莱格等人（1992年），《美国国家科学院院刊》89卷，6911 - 6915页]。此外，相邻的谷氨酸（E954）在所有物种和同工型中都保守，也可能参与阳离子结合。为了进一步探究这些残基在离子转运中的作用，我们采用了诱变 - 表达策略。这种方法避免了向蛋白质中引入大的化学基团，并允许引入特定的氨基酸替换。使用大鼠α - 1 cDNA的定点诱变分别或同时替换大鼠α - 1亚基的谷氨酸残基955和956（对应于猪肾钠钾ATP酶的谷氨酸953和954）。突变cDNA在哇巴因敏感的HeLa细胞中表达。该系统能够快速鉴定出显著损害酶功能的氨基酸替换，因为不影响酶活性的替换在哇巴因存在时会产生菌落，而严重损害功能的替换将阻止或限制哇巴因敏感的HeLa细胞生长。氨基酸替换（E955Q、E956Q、E955Q - E956Q、E955D - E956D）均导致哇巴因敏感细胞生长，表明每种情况下修饰后的钠钾ATP酶都具有功能。为了进一步研究这些改变的酶，分离出抗哇巴因菌落并扩增成稳定的细胞系。（摘要截短于250字）
