Purification of Lipin and Measurement of Phosphatidic Acid Phosphatase Activity from Liposomes.

The lipin family of enzymes are phosphatidic acid (PA) phosphatases responsible for converting PA to diacylglycerol (DAG). Lipins therefore occupy a central node in the synthesis of triacylglycerol (TAG) and phospholipids, and may play a role in regulating the levels of PA and DAG as signaling molecules. Some enzymatic assays used to measure PA phosphatase activities use detergents above their critical micelle concentration to present substrate; however, these methods do not represent the physiological membrane bilayers found in cells and these conditions can drastically alter phosphatase activities. Other assays use poorly defined mixtures of phosphatidylcholine (PC), PA, and high concentrations of BSA to present substrate. In this chapter, we describe methods for affinity purification of FLAG-tagged lipin proteins, and an alternative enzymatic assay using small unilamellar vesicles, also known as liposomes, to investigate specific activities of PA phosphatases. These activities are measured using an acidified Bligh-Dyer extraction to separate the water-soluble, radiolabeled, inorganic phosphate released during the assay from the chloroform-soluble PA.