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Diacylglycerol (DG) lipase, which hydrolyses 1-stearoyl-2-arachidonyl-sn-glycerol to produce an endocannabinoid, 2-arachidonoylglycerol, was purified from the soluble fraction of rat brain lysates. DG lipase was purified about 1,200-fold by a sequential column chromatographic procedure. Among proteins identified by mass spectrometry analysis in the partially purified DG lipase sample, only DDHD domain containing two (DDHD2), which was formerly regarded as a phospholipase A 1 , exhibited significant DG lipase activity. Rat DDHD2 expressed in Chinese hamster ovary cells showed similar enzymatic properties to partially purified DG lipase from rat brain. The source of DG lipase activity in rat brain was immunoprecipitated using anti-DDHD2 antibody. Thus, we concluded that the DG lipase activity in the soluble fraction of rat brain is derived from DDHD2. DDHD2 is distributed widely in the rat brain. Immunohistochemical analysis revealed that DDHD2 is expressed in hippocampal neurons, but not in glia.Keywords: 2-arachidonoylglycerol/diacylglycerol/ endocannabinoid/lipase/phospholipase.Abbreviations: 2-AG, 2-arachidonoylglycerol; CHO, Chinese hamster ovary; CNS, central nervous system; DAB, 3,3 0 -diaminobenzidine; DG, diacylglycerol; DMSO, dimethylsulfoxide; DOC, sodium deoxycholate; DSI, depolarization-induced suppression of inhibition; HA-rDDHD2, HA-tagged rat DDHD2; HRP, horse radish peroxidase; IP, immunoprecipitation; MS/MS, tandem mass spectrometry; PBS, phosphate-buffered saline; PBS-T, PBS containing 0.1% Tween 20; PLA1, phospholipase A 1 ; PLC, phospholipase C; ppt, precipitate; RACE, rapid amplification of cDNA ends; RHC80267, 1,6-bis(cyclohexyloximinocarbonylamino)hexane; SAG, 1-stearoyl-2-arachidonoyl-sn-glycerol; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; sup, supernatant; TG, triacylglycerol; THL, tetrahydrolipstatin; TLC, thin-layer chromatography.Diacylglycerol (DG) lipase is a key enzyme in the pathway of 2-arachidonoylglycerol (2-AG) biosynthesis (1 3). 2-AG, an endocannabinoid, induces various effects by acting on cannabinoid receptors, CB1 and CB2 (1 3). CB1 is highly expressed in the central nervous system (CNS) (4, 5). An important physiological function of 2-AG is the modulation of neurotransmission through CB1 in the CNS. 2-AG has been shown to modulate long-term potentiation, a model for learning and memory (6). Depolarization-induced suppression of inhibition (DSI) is a well-studied electrophysiological process involving endocannabinoids, especially 2-AG (6 8). DSI is the phenomenon whereby post-synaptic depolarization induces inhibition of the release of the neurotransmitter gamma aminobutyric acid. DSI is thought to be one of the mechanisms of short-term synaptic plasticity in the hippocampus.Endocannabinoid signalling also contributes to a wide range of processes including axonal growth and guidance during development, adult neurogenesis and many behavioural responses (9 11).The main pathway for the biosynthesis of 2-AG is thought to be the hydrolysis of arachido...

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