二酰甘油脂肪酶α/β活性测定

Assay of DAGLα/β Activity.

作者信息

Bisogno Tiziana

机构信息

Endocannabinoid Research Group, Institute of Biomolecular Chemistry, National Research Council, Via C. Flegrei 34, 80078, Pozzuoli, Italy.

Center of Integrated Research, Campus Bio-Medico University of Rome, Via Álvaro del Portillo 21, 00128, Rome, Italy.

出版信息

Methods Mol Biol. 2016;1412:149-56. doi: 10.1007/978-1-4939-3539-0_16.

DOI:10.1007/978-1-4939-3539-0_16

PMID:27245901

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.

摘要

内源性大麻素2-花生四烯酸甘油酯（2-AG）通过与1型（CB1）和2型（CB2）大麻素受体结合并使其功能激活来发挥其生理作用。据认为，它是通过sn-1选择性二酰基甘油脂肪酶（DAGL）的作用产生的，该酶催化从含sn-2-花生四烯酸的二酰基甘油生物合成2-AG。由于2-AG生物合成酶直到最近才被鉴定出来，目前关于测量DAGL活性的方法学信息还很少。在此，报道了一种高度灵敏的放射性测定法，该方法使用1-油酰基[1-(14)C]-2-花生四烯酸甘油酯作为底物来测量DAGL活性。详细描述了进行脂质提取、通过薄层色谱（TLC）分离以及通过闪烁计数对放射性标记的[(14)C]-油酸进行定量所需的所有步骤。