Lipid phosphate molecules such as phosphatidate, lysophosphatidate, and diacylglycerol pyrophosphate play roles as signaling molecules in prokaryotic and eukaryotic cells. The cellular processes by which lipid phosphate molecules signal may be attenuated through the action of lipid phosphate phosphatase enzymes. The levels of lipid phosphate phosphatase activities may be used as a marker of signaling events in the cell. In this chapter we describe enzymatic assays that are routinely used to measure the activities of phosphatidate phosphatase, lysophosphatidate phosphatase, and diacylglycerol pyrophosphate phosphatase. These activities are measured by following the release of water-soluble radioactive inorganic phosphate from chloroform-soluble radioactive lipid phosphate substrate following a simple chloroform/methanol/water phase partition.
