Measuring Phosphatidylinositol Generation on Biological Membranes.

Phosphatidylinositol (PI) is a phospholipid molecule required for the generation of seven different phosphoinositide lipids which have a diverse range of signaling and trafficking functions. The precise mechanism of phosphatidylinositol supply during receptor activated signaling and the cellular compartmentation of the synthetic process are still incompletely understood and remain controversial despite several decades of research in this area. The synthesis of phosphatidylinositol requires the activity of an enzyme called phosphatidylinositol synthase, also known as CDIPT, which catalyzes a reversible headgroup exchange reaction on its substrate liponucleotide CDP-diacylglycerol resulting in the incorporation of inositol to generate phosphatidylinositol and the release of CMP. This protocol describes a method for locating PI synthase activity in isolated, intact biological membranes and vesicles.