Advances in Procedures for the Detection and Localization of Inositol Phospholipid Signals in Cells, Tissues, and Enzyme Assays

Publisher Summary This chapter discusses the advances in procedures for the detection and localization of Inositol Phospholipid signals. Inositol phospholipids, collectively known as “phosphoinositides,” comprise of sn-1, 2-diacylglycerol linked at the 3-position of glycerol to the 1-OH of myo-inositol via a phosphodiester bond. The chapter provides a summary on the occurrence, metabolism, and relative abundance of phosphoinositides in mammalian cells. The chapter discusses phosphoinositide-binding protein modules as probes for the sensitive detection of their target lipids in enzyme assays, in cell and tissue extracts and in ultrastructural studies of lipid localization using quantitative immuno-electron microscopy. An advantage of using the targets of signaling lipids in such assay procedures is that their biological functions often require them to display a high degree of selectivity for a specific phosphoinositide species. The chapter describes measurement of signaling lipids as products in enzyme assays and in cell and tissue extracts. These assay procedures exploit the specific lipid binding properties of some of the protein domains listed in one of the tables of the chapter. The chapter also describe specific assays for PtdIns (3, 4, 5) P3 and phosphatidylinositol 3, 4-bisphosphate (PtdIns(3, 4)P2), in cell extracts.