A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase.
Matteo MiceliSilvana CasatiPietro AlleviSilvia BerraRoberta OttriaPaola RotaBruce R BranchiniPierangela CiuffredaPublished in: International journal of molecular sciences (2021)
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.
Keyphrases
- high throughput
- energy transfer
- molecularly imprinted
- air pollution
- cell therapy
- particulate matter
- endothelial cells
- single cell
- induced apoptosis
- hydrogen peroxide
- small molecule
- amino acid
- water soluble
- quantum dots
- dna binding
- polycyclic aromatic hydrocarbons
- risk assessment
- stem cells
- mass spectrometry
- signaling pathway
- pi k akt
- liquid chromatography
- solid phase extraction