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.

A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase / M. Miceli, S. Casati, P. Allevi, S. Berra, R. Ottria, P. Rota, B.R. Branchini, P. Ciuffreda. - In: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. - ISSN 1422-0067. - 22:11(2021 Jun 07). [10.3390/ijms22116148]

A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase

S. Casati
Secondo
;
P. Allevi;S. Berra;R. Ottria;P. Rota;P. Ciuffreda
Ultimo
2021

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.
bioluminescence; monoacylglycerol lipase; kinetic assay; PLG2;
Settore BIO/10 - Biochimica
PIANO DI SOSTEGNO ALLA RICERCA 2015-2017 - LINEA 3 "FINANZIAMENTO PER ACQUISTO/RINNOVO ATTREZZATURE SCIENTIFICHE FINALIZZATE ALLA RICERCA"
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/849076
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