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Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase
Published on: November 23, 2016
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Assay of DAGLα/β Activity
1Endocannabinoid Research Group, Institute of Biomolecular Chemistry, National Research Council, Via C. Flegrei 34, 80078, Pozzuoli, Italy. tbisogno@icb.cnr.it.
Methods in Molecular Biology (Clifton, N.J.)
|June 2, 2016
Summary
This study introduces a new radiometric assay to measure diacylglycerol lipase (DAGL) activity, crucial for understanding 2-arachidonoylglycerol (2-AG) production. The detailed method enables precise quantification of DAGL
Area of Science:
- Biochemistry
- Neuroscience
- Lipid Metabolism
Background:
- The endocannabinoid 2-arachidonoylglycerol (2-AG) modulates physiological functions via CB1 and CB2 receptors.
- 2-AG is synthesized from diacylglycerols by sn-1 selective diacylglycerol lipase (DAGL).
- Limited methodologies exist for measuring DAGL activity due to recent enzyme identification.
Purpose of the Study:
- To develop and detail a sensitive radiometric assay for quantifying DAGL activity.
- To provide a methodological framework for studying 2-AG biosynthesis.
Main Methods:
- Utilized 1-oleoyl[1-(14)C]-2-arachidonoylglycerol as a substrate for DAGL activity measurement.
- Employed lipid extraction and thin-layer chromatography (TLC) for sample preparation.
- Quantified radiolabeled [(14)C]-oleic acid release using scintillation counting.
Main Results:
- A highly sensitive radiometric assay for DAGL activity was successfully established.
- Detailed protocols for lipid extraction, TLC fractionation, and radiometric quantification are provided.
Conclusions:
- The developed radiometric assay offers a robust method for measuring DAGL activity.
- This assay facilitates further research into the biosynthesis and physiological roles of 2-AG.
