Measurements of Phospholipases A2, C, and D (PLA2, PLC, and PLD): In Vitro Microassays, Analysis of Enzyme Isoforms, and Intact-Cell Assays

In order to be properly divisible, the cell membrane has to be remodeled and intracellular membranes must be converted into a vesiculated state prior to mitosis. Phospholipases A₂ , C, and D (PLA₂ , PLC, and PLD) are involved in regulatory events of intracellular mitogen signaling pathways. We describe here three methods for comprehensively assaying those phospholipases: 1) in vitro microassays, in which a radiolabeled substrate is exogenously added to cell lysates to measure the enzyme activity(ies); 2) immunocomplex assays, in which immunoprecipitation with a specific antibody is performed in order to study the contribution of a particular isoform within a family of enzymes; and 3) intact-cell or in vivo assays, in which cells are labeled with a radioactive substrate until steady state is reached. The uniqueness of the in vitro microassay method described here for the first time is that it allows the measurement of, in parallel, the activities of three phospholipases utilizing aliquots derived from the same biological sample. The approach for immunoprecipitation described in this chapter can be extrapolated to the study of a large array of enzyme isoforms. Finally, the intact-cell assays allow for the accurate measurement of receptor-mediated activation in vivo.