Development of a LC-MS/MS based assay for Phosphatidic acid phosphatase activity

SANTIAGO COMBA, SIMÓN MENENDEZ-BRAVO, ANA ARABOLAZA AND HUGO GRAMAJO

Congreso; IX Congreso de Microbiología General; 2013

Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidic acid (PA) yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. These enzymes play a central role in lipid metabolism by directing fatty acid flux to either TAG or phospholipids; and by generating the bioactive lipid DAG involved in intracellular signaling mechanisms. PAP activity has been traditionally assayed by radiochemical analysis, by labeling phosphatidic acid moiety with either 14C, 3H or 32P radioisotopes and measuring the total radioactivity incorporated into the products fraction. Despite its good sensitivity, this method involves the enzymatic synthesis and subsequent purification of the labeled substrate, which makes this technique very tedious and time consuming. Here we have developed a PAP mixed-micelle assay coupled to a LC-MS/MS detection of the DAG product. This method provides excellent detection sensitivity and specificity for the product generated during the reaction, and avoids the need of working with radioisotopes. Furthermore, this protocol can be adapted to detect and characterize the endogenous DAG composition of whole cells extracts.