[25] Measurement of phosphoinositide-specific phospholipase c activity

The chapter presents a study on measurement of phosphoinositide-specific phospholipase C activity. Phosphoinositide-specific phospholipases C (EC 3.1.4.10 and EC 3.1.4.11) comprise a class of phosphodiesterases that catalyze the hydrolysis of either phosphatidylinositol or one of its derivatives to produce diacylglycerol and inositol phosphates. One consequence of the diversity of the phospholipases C is that no single assay is appropriate for all. The methods described in the chapter are restricted to the following conditions: (1) a two-step procedure for purification to near homogeneity of the Mr 86,000 isoform of phospholipase C from human amnion, (2) a general radiochemical procedure for measurement of phospholipase C-catalyzed hydrolysis of phosphoinositides added in suspension, and (3) a method for the measurement of IP3 generated from endogenous substrates by membranes isolated from human polymorphonuclear neutrophils. The method described in the measurement of phospholipase C-catalyzed hydrolysis of phosphoinositides in suspension uses phosphatidyl[2-3H]inositol as substrate but with minor modification and can be employed successfully to measure the rate of hydrolysis of phosphatidyl [2-3H] inositol 4,5-bisphosphate. The chapter describes the isolation of major phospholipase C from human amnion and measurement of phospholipase C-catalyzed production of inositol trisphosphate by membranes from polymorphonuclear neutrophils.