Subtype-specific binding of azidoanilido-GTP by purified G protein alpha subunits.
Subtype-specific binding of azidoanilido-GTP by purified G protein alpha subunits.
复制标题
纯化的 G 蛋白 α 亚基与叠氮苯胺基-GTP 的亚型特异性结合。
DOI：
10.1021/bi00188a017
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Casey,PJ
中科院分区：
文献类型：
--
作者：
Fields,TA;Linder,ME;Casey,PJ
摘要：Azidoanilido-GTP （AA-GTP）是一种抗水解、光反应性的GTP类似物，已成为一种越来越受欢迎的工具，用于鉴定天然质膜内受体对特定G蛋白的激活。尽管使用AA-GTP作为亲和探针，但令人惊讶的是，关于各种G蛋白a亚基结合这种类似物的能力知之甚少。为了直接解决这个问题，我们比较了四种纯化的G蛋白a亚基（Go, Gi2， Gs和Gz）结合AA-GTP的能力与它们结合GTP7S的能力，GTP7S是一种GTP类似物，通常用于表征G蛋白的GTP结合特性。所有四种ga亚单位结合AA-GTP的方式不同于它们结合GTPyS的方式。其中一种蛋白质Gsa需要毫摩尔水平的游离Mg2+才能与AA-GTP显著结合，而Goa和Gia2在大约100毫摩尔水平的游离Mg2+时显示AA-GTP结合的峰值。与GTP7S相比，第四个Ga亚基Gz结合AA-GTP的能力较差，与镁浓度无关。这些结果表明，单个G蛋白a亚基结合AA-GTP的能力存在显著差异。因此，使用AA-GTP来鉴定特定的G蛋白-受体相互作用必须考虑到Ga亚基结合这种类似物的不同能力。
Revised Manuscript Received April 11, 1994® abstract: Azidoanilido-GTP (AA-GTP), a hydrolysis-resistant, photoreactive GTP analog, is becoming an increasingly popular tool for identifying activation of specific G proteins by receptors within native plasma membranes. Despite the use of AA-GTP as an affinity probe, surprisingly little is known regarding the ability of various G protein a subunits to bind this analog. Todirectly address this issue, we compared the ability of four purified G protein a subunits (Go, Gi2, Gs, and Gz) to bind AA-GTP with their ability to bind GTP7S, a GTP analog commonly used to characterize the GTP-binding properties of G proteins. All fourGa subunitstested bound AA-GTP in a manner distinct from their binding of GTPyS. One of these proteins, Gsa, required millimolar levels of free Mg2+ for significant binding of AA-GTP, while Goa and Gia2 displayed peak AA-GTP binding at approximately 100 pM free Mg2+. The fourth Ga subunit, Gz, bound AA-GTP very poorly relative to GTP7S regardless of the magnesium concentration. These results indicatethat individual G protein a subunits differ markedlyin their ability to bind AA-GTP. Use of AA-GTP to identify specific G protein-receptor interactions must therefore take into account the varied abilities of Ga subunitsto bind this analog.