Van Huysse J W, Lingrel J B
Dept. of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, OH 45267-0524.
Cell Mol Biol Res. 1993;39(5):497-507.
Side chain oxygens are critical in the binding of cations by several macromolecules, and chemical labeling studies suggest that the carboxyl oxygens of E953 of the pig kidney Na, K-ATPase are essential for Na+ and K+ ligation. An adjacent residue, E954, is highly conserved and may also be important in cation binding. Replacement of the corresponding glutamates in the rat alpha 1 isoform (E955 and E956) with glutamines or aspartic acids has only a modest effect on enzyme activity, but both substituted amino acids contain at least one side chain oxygen, and may fulfill the role of the wild-type glutamates in binding cations. Since substitutions of amino acids lacking an oxygenated side chain have not been made at positions 955 and 956, nonpolar amino acid replacements (E955A, E956A, E955A-E956A, and E955L-E956L) were made, and the effects of the substitutions on the cation dependence properties of the mutant enzymes were examined. The substitutions were made using site-directed mutagenesis of the rat alpha 1 cDNA (which encodes a ouabain-resistant alpha subunit), followed by transfection of wild-type and mutant cDNAs into ouabain-sensitive HeLa cells, with subsequent expression of the altered proteins. Transfection with all cDNA constructs resulted in the ouabain resistance of transfected HeLa cells, demonstrating that the modified Na, K-ATPase in each case was functional. Ouabain resistance of Na, K-ATPase activity was increased 1,000-fold in microsomal membranes isolated from cells transfected with wild-type rat alpha 1 (WT) and all mutant cDNA constructs, compared to activity in membranes prepared from untransfected HeLa cells. This confirmed the expression of a ouabain-resistant enzyme in all transfected cell lines, and the enhanced ouabain resistance permitted the study of the exogenous, expressed Na, K-ATPase in the presence of 5.0 microM ouabain. Cation stimulation of exogenous Na, K-ATPase activity was not affected by the E955A substitution and only slightly by the E956A replacement (K0.5(Na+) of 3.40 +/- 0.21, 3.30 +/- 0.22, and 6.60 +/- 0.55 mM NaCl for WT, E955A and E956A, respectively; K0.5(K+) of 0.78 +/- 0.01, 0.74 +/- 0.10, and 0.56 +/- 0.11 mM KCl). Even doubly substituted enzymes had only mild alterations in cation dependence properties (K0.5(Na+) of 7.54 +/- 0.23, 7.14 +/- 0.10 mM NaCl for E955A-E956A, E955L-E956L, respectively; K0.5(K+) of 0.43 +/- 0.13, 1.83 +/- 0.13 mM KCl). The results demonstrate that there are no requirements for an oxygenated side chain at positions 955 and 956 for normal or nearly normal cation dependence.
侧链氧原子在几种大分子与阳离子的结合中至关重要，化学标记研究表明猪肾钠钾ATP酶E953的羧基氧原子对于钠离子和钾离子的连接必不可少。相邻残基E954高度保守，在阳离子结合中可能也很重要。用谷氨酰胺或天冬氨酸取代大鼠α1同工型（E955和E956）中的相应谷氨酸对酶活性只有适度影响，但这两种取代氨基酸都至少含有一个侧链氧原子，可能在结合阳离子方面发挥野生型谷氨酸的作用。由于尚未在955和956位进行缺乏氧化侧链氨基酸的取代，因此进行了非极性氨基酸取代（E955A、E956A、E955A - E956A和E955L - E956L），并检测了这些取代对突变酶阳离子依赖性特性的影响。使用大鼠α1 cDNA（编码对哇巴因耐药的α亚基）的定点诱变进行取代，随后将野生型和突变型cDNA转染到对哇巴因敏感的HeLa细胞中，随后表达改变后的蛋白质。用所有cDNA构建体转染导致转染的HeLa细胞对哇巴因耐药，表明每种情况下修饰后的钠钾ATP酶都具有功能。与未转染的HeLa细胞制备的膜中的活性相比，从用野生型大鼠α1（WT）和所有突变型cDNA构建体转染的细胞中分离的微粒体膜中，钠钾ATP酶活性的哇巴因耐药性增加了1000倍。这证实了所有转染细胞系中都表达了对哇巴因耐药的酶，增强的哇巴因耐药性使得能够在存在5.0微摩尔哇巴因的情况下研究外源表达的钠钾ATP酶。E955A取代对外源钠钾ATP酶活性的阳离子刺激没有影响，E956A取代仅有轻微影响（WT、E955A和E956A的NaCl的K0.5分别为3.40±0.21、3.30±0.22和6.60±0.55毫摩尔；KCl的K0.5分别为0.78±0.01、0.74±0.10和0.56±0.11毫摩尔）。即使是双取代酶在阳离子依赖性特性方面也只有轻微改变（E955A - E956A、E955L - E956L的NaCl的K0.5分别为7.54±0.23、7.14±0.10毫摩尔；KCl的K0.5分别为0.43±0.13、1.83±0.13毫摩尔）。结果表明，955和956位对于正常或接近正常的阳离子依赖性不需要氧化侧链。
