FLUORESCENCE-BASED ASSAY FOR MONOACYLGLYCEROL LIPASE COMPATIBLE WITH INHIBITOR SCREENING
申请人:Chanda Pranab Kumar
公开号:US20090311723A1
公开(公告)日:2009-12-17
The present invention provides reagents, kits and methods for assaying monoglycerol lipase activity and for identifying compounds that modulate monoglycerol lipase (“MGL”) activity. A simple, sensitive fluorescence assay, which is amenable to high throughput screening, is described. In one embodiment, 7-Hydroxycoumarinyl-arachidonate (7-HCA) is used as a fluorogenic substrate for MGL, which catalyzes the hydrolysis of 7-HCA to generate arachidonic acid and the highly fluorescent 7-hydroxycoumarin (7-HC). Release of 7-HC is monitored continuously using a fluorometer. MGL protein catalyzed the hydrolysis of 7-HCA with an apparent KM of 9.8 mM and Vmax of 1.7 mmoles min
−1
mg protein
−1
.