A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase
Abstract
A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Jun 07, 2021
- Source ID
- 10.3390/ijms22116148
Entities
People
- Bruce R Branchini
- Matteo Miceli
- Paola Rota
- Pierangela Ciuffreda
- Pietro Allevi
- Roberta Ottria
- Silvana Casati
- Silvia Berra
Organizations
- Air Force Office of Scientific Research
- University of Milan