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Structure and function of ion-channel VO of Na+-translocating V-ATPase

钠转运V-ATP酶离子通道VO的结构与功能

基本信息

批准号：
13672272
负责人：
KAKINUMA Yoshimi
金额：
$ 2.62万
依托单位：
MURORAN INSTITUTE OF TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672272/
关键词：
V-ATPase Sodium Ion Enterococcus hirae

项目摘要

The role of NtpI C-terminal region of E. hirae Na^+-ATPase was studied. In this region, there is an arginine residue, which is highly conserved among the corresponding subunits of bacterial V-ATPases, at position 573 of NtpI. Substitution of Arg-573 with Glu, Leu, or Gln abolished sodium transport and sodium-stimulated ATP hydrolysis of the enzyme. The conservative replacement of Arg by Lys lowered both activities to about one fifth of those of the wild-type enzyme. We previously reported an ATP-dependent negative cooperativity for Na^+ coupling of this enzyme. This negative cooperativity was weakened by the mutation Arg573Lys; the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22±0.03 and 0.40±0.05, respectively. The Hill coefficients of both enzymes at limited ATP concentrations approached 1. These results indicate that NtpI Arg573 is indispensable for sodium translocation and for the cooperative features of E. hirae V-ATPase.In addition to Arg-573, the Tyr571, Leu574 and Leu577 were also involved in Na^+ translocation. And other important residues, His-626 and Glu-634, were also required for Na^+-ATPase activity, but their role in ion translocation may differ from conserved His or Glu residues of other a subunits. From these results, it is indicated that the C-terminal region of NtpI is both essential in sodium translocation and stabilization of NtpI.

研究了海蝇Na^+- atp酶c端NtpI的作用。在这个区域，有一个精氨酸残基，在细菌V-ATPases的相应亚基中高度保守，位于NtpI的573位。用谷氨酸、亮氨酸或谷氨酸取代精氨酸-573可消除钠转运，并可刺激酶的ATP水解。赖氨酸保守替代Arg后，两种酶的活性均降至野生型酶的五分之一左右。我们之前报道了该酶对Na^+偶联的atp依赖性负协同性。突变Arg573Lys削弱了这种负协同性；在饱和ATP浓度下，野生型和突变型酶的Hill系数分别为0.22±0.03和0.40±0.05。在有限ATP浓度下，两种酶的Hill系数均接近1。这些结果表明，NtpI Arg573在钠转运和E. hirae V-ATPase的协同特性中是不可或缺的。除Arg-573外，Tyr571、Leu574和Leu577也参与Na^+易位。而其他重要残基His-626和Glu-634也是Na^+- atp酶活性所必需的，但它们在离子转运中的作用可能不同于其他a亚基的保守的His或Glu残基。这些结果表明，NtpI的c端区域在钠转运和NtpI的稳定中都是必不可少的。