Conversion of the Low Affinity Ouabain-binding Site of Non-gastric H,K-ATPase into a High Affinity Binding Site by Substitution of Only Five Amino Acids*
Conversion of the Low Affinity Ouabain-binding Site of Non-gastric H,K-ATPase into a High Affinity Binding Site by Substitution of Only Five Amino Acids*
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通过仅取代 5 个氨基酸，将非胃 H,K-ATP 酶的低亲和力哇巴因结合位点转化为高亲和力结合位点*
DOI：
10.1074/jbc.m600551200
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发表时间：
2006
影响因子：
4.8
通讯作者：
J. De Pont
中科院分区：
文献类型：
--
作者：
L. Qiu;H. Swarts;E. Tonk;P. Willems;J. B. Koenderink;J. De Pont
IIC亚家族的P型ATPase对哇巴因的敏感性有很大差异。这允许一种策略，其中该亚家族的哇巴因不敏感成员被用作突变阐明哇巴因结合位点的模板。通过这一策略，我们最近在Na，K-ATPase中发现了七个氨基酸，它们与胃H，K-ATPase具有高亲和力的哇巴因结合(Chu，L.Y.，Krieger，E.，Schaftenaar，G.，Swarts，H.G.P.，Willems，P.H.G.M.，de Pont，J.J.H.H.M.和Koenderink，J.B.(2005)J.Biol)。化学。280、32349-32355)。因为重要但相同的氨基酸在那项研究中没有被识别，在这里我们使用非胃H，K-ATPase，它对哇巴因相当不敏感，作为模板。该酶的催化亚基与Na，K-ATPaseβ1亚基一起在非洲爪哇卵母细胞中表达，其中含有Na，K-ATP酶的多种氨基酸。一个含有14个氨基酸的嵌合体，位于Na，K-ATPase特有的M4、M5和M6中，显示出高亲和力的哇巴因结合。其中四个残基都位于M5，对于高亲和力结合似乎是不必要的。将其余10个残基单独突变为非胃H，K-ATPase对应的氨基酸，产生了5个氨基酸(Glu312，Gly319，Pro778，Leu795和Cys802)，它们的突变导致哇巴因结合丧失。在最后的功能增益实验中，我们在非胃H，K-ATPase中以不同的组合引入了这五种氨基酸，并证明了这五种氨基酸都是高亲和力哇巴因结合所必需的。具有这5个突变的非胃H，K-ATPase对哇巴因的表观亲和力与野生型Na，K-ATPase相似，对哇巴因的亲和力是野生型Na，K-ATPase的2000倍。
P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349–32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase β1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document}-stimulated ATPase activity in membranes of transfected Sf9 cells.
影响因子：
2.9
作者：
FENG, JN;LINGREL, JB
通讯作者：
LINGREL, JB
编码假定远端结肠 H ,K( )-ATP 酶的 cDNA 的分离和表征。
DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Crowson,MS;Shull,GE
通讯作者：
Shull,GE
影响因子：
5.6
作者：
HENDERSON, R;BALDWIN, JM;DOWNING, KH
通讯作者：
DOWNING, KH