Measurement of phosphoinositide 3-kinase products in cultured Mammalian cells by HPLC.
The phosphoinositide 3-kinase (PI3K) family catalyses the addition of a phosphate group to the D-3 position of polyphosphoinositides (PPIn). Since the discovery in the late 80s that phosphatidylinositol is phosphorylated in the D-3 position in eukaryotic cells, there has been an explosion of interest in these PPIn. Although the four D-3 PPIn (phosphatidylinositol 3-phophate (PtdIns3P), PtdIns(3,4)P(2), PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3)) represent only a small proportion of PPIn, production of D-3 PPIn is required for an ever-increasing number of processes. Measurement of the PPIn levels in intact cells cultured cells has been vital to our understanding of the metabolism and function of these important signalling molecules; methods are described herein that allow measurement of PPIn levels in cultured cells, with emphasis on the 3-OH PPIn.
Cooke
Frank T
FT
Department of Structural and Molecular Biology, University College London, London, UK. f.cooke@ucl.ac.uk
D000818:Animals; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D010716:Phosphatidylinositols; D019869:Phosphatidylinositol 3-Kinases