2-Azido-[32P]NAD+, a photoactivatable probe for G-protein structure: evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of alpha interacts with both alpha and gamma subunits.
2-Azido-[32P]NAD+, a photoactivatable probe for G-protein structure: evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of alpha interacts with both alpha and gamma subunits.
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2-叠氮基-[32P]NAD，一种用于 G 蛋白结构的光激活探针：全转导蛋白寡聚物的证据，其中 α 的 ADP 核糖基化羧基末端与 α 和 γ 亚基相互作用。
DOI：
10.1073/pnas.87.10.3645
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发表时间：
1990
影响因子：
11.1
通讯作者：
Ruoho,AE
中科院分区：
文献类型：
--
作者：
Vaillancourt,RR;Dhanasekaran,N;Johnson,GL;Ruoho,AE
合成了一种NAD+的放射性光活化衍生物，2-氮基-[腺苷- 32p]NAD+，并与百日毒一起用于视网膜鸟嘌呤核苷酸结合蛋白GT α亚基（α T）的adp -核糖化Cys347。adp -核糖基化后，2-叠氮-[腺苷- 32p] adp核糖的叠氮化部分被光激活，产生涉及GT异三聚体α和γ亚基的四种交联物质：α三聚体（α - α - α）和α - α - γ交联，α二聚体（α - α）和α - γ交联。α三聚体、α - α - γ复合物、α二聚体和α - γ复合物对α T抗体具有免疫反应性。α - α - γ和α - γ复合物与识别γ亚基的抗血清具有免疫反应性。没有发现T与T亚基交联的证据。使用醋酸汞水解Cys347和2-叠氮-[腺苷- 32p] adp核糖之间的硫糖苷键，导致α T的放射性标记从交联低聚物中的Cys347转移到α单体，表明分子间光交联，并转移到γ单体，表明分子间交联复合物（在异三聚体之间）或分子内交联复合物（在异三聚体内部）。这些结果表明GT以低聚物的形式存在，并且adp核糖化的Cys347（距离α t -羧基端4个残基）指向并靠近γ亚基。
A radioactive and photoactivatable derivative of NAD+, 2-azido-[adenylate-32P]NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the alpha subunit (alpha T) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of alpha T followed by light activation of the azide moiety of 2-azido-[adenylate-32P]ADP-ribose produced four crosslinked species involving the alpha and gamma subunits of the GT heterotrimer: an alpha trimer (alpha-alpha-alpha), and alpha-alpha-gamma crosslink, an alpha dimer (alpha-alpha), and an alpha-gamma crosslink. The alpha trimer, alpha-alpha-gamma complex, alpha dimer, and alpha-gamma complexes were immunoreactive with alpha T antibodies. The alpha-alpha-gamma and the alpha-gamma complexes were immunoreactive with antisera recognizing gamma subunits. No evidence was found for crosslinking of alpha T to beta T subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-[adenylate-32P]ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of alpha T in the crosslinked oligomers to alpha monomers, indicative of intermolecular photocrosslinking, and to gamma monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the alpha T-carboxyl terminus, is oriented toward and in close proximity to the gamma subunit.