Losito Oriana, Szijgyarto Zsolt, Resnick Adam Cain, Saiardi Adolfo
Department of Cell and Developmental Biology, Medical Research Council Cell Biology Unit and Laboratory for Molecular Cell Biology, University College London, London, United States of America.
PLoS One. 2009;4(5):e5580. doi: 10.1371/journal.pone.0005580. Epub 2009 May 18.
Inositol pyrophosphates are a recently characterized cell signalling molecules responsible for the pyrophosphorylation of protein substrates. Though likely involved in a wide range of cellular functions, the study of inositol pyrophosphates has suffered from a lack of readily available methods for their analysis.
We describe a novel, sensitive and rapid polyacrylamide gel electrophoresis (PAGE)-based method for the analysis of inositol pyrophosphates. Using 4',6-diamidino-2-phenylindole (DAPI) and Toluidine Blue we demonstrate the unequivocal detection of various inositol pyrophosphate species.
The use of the PAGE-based method reveals the likely underestimation of inositol pyrophosphates and their signalling contribution in cells when measured via traditional HPLC-based techniques. PAGE-based analyses also reveals the existence of a number of additional, previously uncharacterised pyrophosphorylated inositol reaction products, defining a more complex metabolism associated with the catalytically flexible kinase class responsible for the production of these highly energetic cell signalling molecules.
肌醇焦磷酸是最近被鉴定出的细胞信号分子，负责蛋白质底物的焦磷酸化。尽管可能参与多种细胞功能，但对肌醇焦磷酸的研究一直因缺乏易于使用的分析方法而受到阻碍。
我们描述了一种基于聚丙烯酰胺凝胶电泳（PAGE）的新颖、灵敏且快速的肌醇焦磷酸分析方法。使用4',6-二脒基-2-苯基吲哚（DAPI）和甲苯胺蓝，我们明确检测到了各种肌醇焦磷酸物种。
基于PAGE的方法表明，当通过传统的基于高效液相色谱（HPLC）的技术进行测量时，细胞中肌醇焦磷酸及其信号贡献可能被低估。基于PAGE的分析还揭示了许多额外的、以前未被表征的焦磷酸化肌醇反应产物的存在，这定义了一种与负责产生这些高能细胞信号分子的催化灵活激酶类别相关的更复杂代谢。
