Monitoring Inositol-Specific Phospholipase C Activity Using a Phospholipid FlashPlate®

Inositol-specific phospholipase Cs(PLCs) are a group of enzymes involved in the signal transduction pathway of many plasma membrane receptor mediated events. We developed a modified solid surface to capture [3H] PIP2 onto the Basic FlashPlate® in order to monitor PLC activity. Our results clearly demonstrate the utility of [3H] PIP2-Coated Phospholipid FlashPlate® microtiter plates for assessing PLC activity for HTS of receptor-coupled functional assays. The results show that PLC activity can be measured easily from a variety of sources including purified recombinant enzyme preparations, crude HL60 cell lysates and permeabilized A431 human carcinoma cells. Moreover, this format provides a surface comparable to that used for classical solution based radiolabeled mixed phospholipid micelle studies and illustrates the feasibility of this assay for measuring PLC activation in a variety of different drug screening assays.