title: Assay of DAGLα/β Activity.
abstract: The endocannabinoid 2-arachidonoylglycerol (2-AG) exerts its physiological action by binding to and functionally activating type-1 (CB1) and type-2 (CB2) cannabinoid receptors. It is thought to be produced through the action of sn-1 selective diacylglycerol lipase (DAGL) that catalyzes 2-AG biosynthesis from sn-2-arachidonate-containing diacylglycerols. Since 2-AG biosynthetic enzymes have been identified only recently, little information on methodological approaches for measuring DAGL activity is as yet available. Here, a highly sensitive radiometric assay to measure DAGL activity by using 1-oleoyl[1-(14)C]-2-arachidonoylglycerol as the substrate is reported. All the steps needed to perform lipid extraction, fractionation by thin-layer chromatography (TLC), and quantification of radiolabeled [(14)C]-oleic acid via scintillation counting are described in detail.
authors.affiliation: Endocannabinoid Research Group, Institute of Biomolecular Chemistry, National Research Council, Via C. Flegrei 34, 80078, Pozzuoli, Italy. tbisogno@icb.cnr.it.|Center of Integrated Research, Campus Bio-Medico University of Rome, Via Álvaro del Portillo 21, 00128, Rome, Italy. tbisogno@icb.cnr.it.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Bisogno
mesh_terms: D001095:Arachidonic Acids / Q000096:biosynthesis; D002855:Chromatography, Thin Layer; D063388:Endocannabinoids / Q000096:biosynthesis; D057075:Enzyme Assays; D005989:Glycerides / Q000096:biosynthesis; D008071:Lipoprotein Lipase / Q000378:metabolism*; D011874:Radiometry / Q000379:methods
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; D005989:Glycerides; C094503:glyceryl 2-arachidonate; D008071:Lipoprotein Lipase
keywords: 2-Arachidonoylglycerol; Diacylglycerol lipase; Radiometric assay; Thin-layer chromatography