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KMID : 0603820100160040297
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Journal of Experimental & Biomedical Science
2010 Volume.16 No. 4 p.297 ~ p.285
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Fluorescence-based Assay System for Endocannabinoid Degradation Enzyme, Fatty Acid Amide Hydrolase
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Kim Dae-Woong
Kim Gun-Joong
Kim Hae-Jo
Ghil Sung-Ho
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Abstract
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Endogenous cannabinoids (endocannabinoids) display various pharmacological effects including pain control, anti-inflammation, and neuroprotection. The synthesis and release of endocannabinoids are regulated under both physiological and pathological conditions. The main degrading enzyme of endocannabinoid is fatty acid amide hydrolase (FAAH). Therefore we have developed the fluorescence-based assay system for FAAH. We established stable CosM6 cell lines expressing human FAAH. We also synthesized 2-oxo-2H-chromen-7-yl decanoate (DAEC) as a fluorogenic substrate for FAAH. When crude membrane extracts stably expressing FAAH was incubated with DAEC at 25℃, FAAH reacted specifically to DAEC and catalyzes the hydrolysis of DAEC into decanoic acid and highly fluorescent coumarin. Furthermore, the serin hydrolase inhibitor, phenylmethanesulfonylfluoride, inhibited the coumarin release to the reaction buffer in concentration dependent manner. This assay system is suitable for high-throughput screening since this system has simple experimental procedure and measurement method.
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KEYWORD
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Fatty acid amide hydrolase, Cannabinoid, High throughput screening, Fluorescent assay, Enzymatic assay, Esterase
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