Abstract
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is an enzyme that converts phosphatidylinositol 4-phosphate [PI4P] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. 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 [P32] ATP into PI(4,5)P2 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.
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Acknowledgments
I thank Christopher J. Stefan for giving me a great opportunity to write this method article. This new technique I have shown in this chapter has been established when I had worked as a postdoctoral researcher in the Stefan lab at UCL. I thank members of the Tooze lab at the Crick Institute for helpful discussions. In particular, Javier H. Hervás gave helpful suggestions regarding liposome preparation. Taki Nishimura was supported by JSPS Postdoctoral Fellowships for Research Abroad and the Osamu Hayaishi Scholarship for Study Abroad.
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Nishimura, T. (2021). A Real-Time Phosphatidylinositol 4-Phosphate 5-Kinase Assay Using Fluorescence Spectroscopy. In: Botelho, R.J. (eds) Phosphoinositides. Methods in Molecular Biology, vol 2251. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1142-5_8
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DOI: https://doi.org/10.1007/978-1-0716-1142-5_8
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