Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins.
Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins.
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GTP 结合蛋白上的核苷二磷酸激酶将 GDP 转化为 GTP。
DOI：
10.1016/s0021-9258(18)45773-0
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
T. Katada
中科院分区：
文献类型：
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作者：
Satoshi Kikkawa;K.;N. Shimada;Michio Ui;Narimichi Kimura;T. Katada
使用均质纯化的蛋白质研究了 alpha beta gamma 三聚体 GTP 结合蛋白（G 蛋白；G0 和 Gs）与核苷二磷酸激酶（NDP 激酶）的直接相互作用。当 GDP 结合的 G0 与 NDP 激酶一起孵育时，[gamma-32P]ATP 逐渐释放 32Pi。 G0 与 NDP 激酶相互作用诱导的 Pi 释放并不伴随与 G0 α 亚基结合的 GDP 解离。这与以 GTP 为底物观察到的 G 蛋白催化 GTP 水解形成鲜明对比；结合的 GDP 的解离对于后续底物 GTP 的结合进行水解是必需的。动力学分析显示游离 GDP 和 G 蛋白结合 GDP 之间 NDP 激酶底物的不同特性。使用 5'-(3-O-硫代)三磷酸腺苷作为磷酸供体，确实证明了 G0 上 GDP 的 NDP 激酶依赖性磷酸化； ATP 类似物形成鸟苷 5'-(3-O-硫代)三磷酸结合的 G0。此外，在NDP激酶、ATP和ADP核糖基化因子存在下，纯化的Gs很容易被霍乱毒素ADP核糖基化，这也表明Gs上的核苷酸形式肯定是GTP。这些结果表明NDP激酶可以将ATP的γ-磷酸直接转移至与G蛋白结合的GDP，并且这种磷酸化导致信号偶联蛋白的激活。与之前描述的激动剂-受体复合物的 GDP-GTP 交换途径相比，讨论了 G 蛋白新激活机制的可能作用。
A direct interaction of alpha beta gamma trimeric GTP binding proteins (G proteins; G0 and Gs) with nucleoside diphosphate kinase (NDP kinase) was investigated with homogeneously purified proteins. There was a progressive release of 32Pi from [gamma-32P]ATP when GDP-bound G0 was incubated together with NDP kinase. The Pi release induced by the interaction of G0 with NDP kinase was not accompanied by the dissociation of GDP bound to the alpha-subunit of G0. This was a sharp contrast to G protein-catalyzed GTP hydrolysis observed with GTP as the substrate; the dissociation of bound GDP was essentially required for the following binding of the substrate, GTP, to be hydrolyzed. A kinetic analysis displayed different properties for the substrate of NDP kinase between free GDP and G protein-bound GDP. NDP kinase-dependent phosphorylation of GDP on G0 was indeed demonstrated with adenosine 5'-(3-O-thio)triphosphate as the phosphate donor; there was a formation of guanosine 5'-(3-O-thio)triphosphate-bound G0 from the ATP analogue. Moreover, purified Gs was readily ADP-ribosylated by cholera toxin in the presence of NDP kinase, ATP, and an ADP-ribosylation factor, also suggesting that the nucleotide form on Gs was certainly GTP. These results indicate that NDP kinase can transfer the gamma-phosphate of ATP directly to GDP bound to G proteins and that this phosphorylation results in the activation of the signal-coupling proteins. A possible role of the new activation mechanism of G proteins is discussed in comparison with the previously characterized GDP-GTP exchange pathway by the agonist-receptor complex.