利用荧光光谱法进行实时磷脂酰肌醇 4,5-二磷酸激酶分析。
A Real-Time Phosphatidylinositol 4-Phosphate 5-Kinase Assay Using Fluorescence Spectroscopy.
机构信息
MRC Laboratory for Molecular Cell Biology, University College London, London, UK.
Molecular Cell Biology of Autophagy, The Francis Crick Institute, London, UK.
出版信息
Methods Mol Biol. 2021;2251:121-132. doi: 10.1007/978-1-0716-1142-5_8.
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is an enzyme that converts phosphatidylinositol 4-phosphate [PI4P] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P]. PIP5K plays a key role in the regulation of vesicular transport, cytoskeleton reorganization, and cell division. In general, to investigate an enzymatic activity of PIP5K, the amount of incorporated [P] ATP into PI(4,5)P fraction is measured in in vitro reconstitution experiments. However, tools to monitor dynamic changes in its activity in real time have been lacking. Recently, we have developed a novel PIP5K assay using fluorescence spectroscopy. Compared to conventional methods in which lipids extraction steps are needed, our method is easy and quick to perform and enables a real-time analysis. This chapter provides a protocol to set up and perform the novel PIP5K assay we have recently established.
磷脂酰肌醇 4-磷酸 5-激酶（PIP5K）是一种将磷脂酰肌醇 4-磷酸 [PI4P] 转化为磷脂酰肌醇 4,5-二磷酸 [PI(4,5)P] 的酶。PIP5K 在囊泡运输、细胞骨架重组和细胞分裂的调节中起着关键作用。通常，为了研究 PIP5K 的酶活性，在体外重组实验中测量 [P]ATP 掺入到 PI(4,5)P 馏分中的量。然而，缺乏实时监测其活性的动态变化的工具。最近，我们使用荧光光谱法开发了一种新型的 PIP5K 测定法。与需要脂质提取步骤的传统方法相比，我们的方法易于操作且快速，并且能够进行实时分析。本章提供了建立和执行我们最近建立的新型 PIP5K 测定法的方案。
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