Chapter title |
Assay of DAGLα/β Activity
|
---|---|
Chapter number | 16 |
Book title |
Endocannabinoid Signaling
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3539-0_16 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3537-6, 978-1-4939-3539-0
|
Authors |
Tiziana Bisogno |
Editors |
Mauro Maccarrone |
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. |
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