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首页> 外文期刊>Biochemistry >Evidence for disaggregation of oligomeric G_oalpha induced by guanosine-5'-3-O-(thio)triphosphate activation
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Evidence for disaggregation of oligomeric G_oalpha induced by guanosine-5'-3-O-(thio)triphosphate activation

机译：鸟苷5'-3-O-（硫代）三磷酸活化引起的寡聚G_oalpha分解的证据

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摘要

Myristoylated G_oalpha was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60(G_oalpha) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G_0alpha, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) activation induced disaggregation of the G_oalpha oligomers to monomers. The G_oalpha was crosslinked by a cross-linked, N,N'-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products. In contrast, no obvious cross-linking occurred when G_oalpha was pretreated with GTPgammaS. Immunoblot analysis also demonstrated oligomerization of the purified G_oalpha proteins and its disaggregation triggered by GTPgammaS. These results provided direct evidence for the "disaggregation-coupling" theory and the disaggregation action of GTPgammaS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.
机译：在与pQE60（G_oalpha）和pBB131（N-肉豆蔻酰基转移酶，NMT）共转化的大肠杆菌菌株JM109中表达并高度纯化了肉豆蔻酰基化的G_oalpha。非变性凝胶电泳和凝胶过滤分析表明，G_0alpha以GDP结合形式可形成涉及二聚体，三聚体，四聚体，五聚体或六聚体的寡聚体和鸟苷5'-3-O-（硫代）三磷酸酯（GTPgammaS ）激活导致G_oalpha低聚物解聚为单体。 G_oalpha通过交联的N，N'-1,4-苯二甲基亚胺（p-PDM）交联，得到多个交联产物。相反，当用GTPgammaS预处理G_oalpha时，没有发生明显的交联。免疫印迹分析还证明了纯化的G_oalpha蛋白的寡聚及其由GTPgammaS触发的分解。这些结果为“分解-偶联”理论提供了直接的证据，GTPgammaS的分解作用可能进一步阐明GDP / GTP交换在G蛋白偶联信号转导途径中的调控作用。

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