title: Enzymatic characterization of recombinant rat DDHD2: a soluble diacylglycerol lipase.
abstract: DDHD2 has been reported to exhibit phospholipase A1, triacylglycerol (TG) lipase and diacylglycerol (DG) lipase activities. However, the detailed enzymatic properties of DDHD2 have not yet been elucidated. In the current study, the substrate specificity of DDHD2 towards DG, TG and phosphatidic acid (PA) has been examined using highly purified recombinant rat DDHD2 (rDDHD2) with a liquid chromatography mass spectrometer. The kcat/Kmvalue for DG (18:0/20:4) was much higher than those for TG (18:1/18:1/18:1), and PA (18:0/20:4) in the presence of sodium deoxycholate. The enzyme activity of rDDHD2 towards DG (18:0/20:4) was highest among all of the substrates tested. In addition, rDDHD2 was highly specific to DG substrates with a polyunsaturated fatty acid at their sn-2 position. The levels of 2-arachidonoylglycerol (2-AG) in CHO cells were quantified by gas chromatography-tandem mass spectrometry, showing that CHO cells expressing recombinant rDDHD2 contained higher levels of 2-AG when cells were treated with a monoacylglycerol lipase inhibitor, URB602. These results therefore support the idea that DDHD2 functions as a DG lipase in vivo and produces 2-AG.
authors.affiliation: Department of Biochemistry.|Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Araki
authors.affiliation: Department of Biochemistry ohshima-tky@umin.ac.jp.
authors.forename: Noriyasu
authors.initials: N
authors.lastname: Ohshima
authors.affiliation: National Hospital Organization Takasaki General Medical Center, Takasaki, Gunma 370-0829, Japan.
authors.forename: Chizu
authors.initials: C
authors.lastname: Aso
authors.affiliation: Department of Biochemistry.
authors.forename: Akimitsu
authors.initials: A
authors.lastname: Konishi
authors.affiliation: Department of Biochemistry.
authors.forename: Hideru
authors.initials: H
authors.lastname: Obinata
authors.affiliation: Department of Biochemistry.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Tatei
authors.affiliation: Department of Biochemistry.
authors.forename: Takashi
authors.initials: T
authors.lastname: Izumi
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry* / Q000378:metabolism*; D001713:Biphenyl Compounds / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D063388:Endocannabinoids / Q000737:chemistry* / Q000378:metabolism*; D005989:Glycerides / Q000737:chemistry* / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects / Q000502:physiology
chemical_list: D001095:Arachidonic Acids; D001713:Biphenyl Compounds; D063388:Endocannabinoids; D005989:Glycerides; D011994:Recombinant Proteins; C501835:URB602; C094503:glyceryl 2-arachidonate; D008071:Lipoprotein Lipase
keywords: 2-arachidonoylglycerol; DDHD2; diacylglycerol; lipase; phospholipase