Measurement of Subcellular Sites of Polyphosphoinositide Metabolism in Isolated Rat Hepatocytes

The products of phosphatidylinositol bisphosphate (PIP₂) degradation––diacylglycerol and D-myo-inositol 1, 4, 5-trisphosphate (IP₃)––have both been identified as intracellular second messengers. Numerous cell types respond to a variety of stimuli with the enhanced hydrolysis of PIP₂ and to a lesser extent of phosphatidylinositol 4-phosphate (PIP). The plasma membrane PIP₂ phosphodiesterase does not appear to hydrolyze phosphatidylinositol (PI) in liver. One approach to the investigation of the subcellular location of polyphosphoinositide metabolic reactions is to fractionate cell homogenates by the best procedures available and to assay the respective enzymes with added substrates under V_max conditions. A second approach for determining the subcellular sites of the metabolism of PIP and PIP₂ is to analyze the distribution of PIP and PIP₂, the products of PI and PIP kinase, in a cell; however, their low levels make analysis by existing chemical means virtually impossible. The chapter explains the preparation of phosphatidylinositol [4-³²P] phosphate and phosphatidylinositol [4,5-³²p]bisphosphate.