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KMID : 0603820120180040438
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Journal of Experimental & Biomedical Science
2012 Volume.18 No. 4 p.438 ~ p.444
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Assay System for N-acylethanolamines Degradation Enzyme, N-acylethanolamine-hydrolyzing Acid Amidase
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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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N-acylethanolamines (NAEs) including endocannabinoids, anadamide, are long chain fatty acid ethanolamines and express ubiquitously in animal and plant tissues. NAEs have several pharmacological effects including anti-inflammatory, analgesic and anorexic effects. The levels of NAEs in tissues are strictly regulated by synthesizing and hydrolyzing enzymes because NAEs are not stored in the cell but rather made on demand. NAEs are hydrolyzed to free fatty acids and ethanolamines by fatty acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase (NAAA). Here, we suggest the fluorescence-based assay system for NAAA. We developed N-(4-methy-2-oxo-2H-chromen-7-yl)palmitamide (PAAC) as a fluorogenic substrate for NAAA and we also generated NAAA stably expressing COSM6 cell line. When extracts of cells expressing NAAA were incubated with PAAC, NAAA specifically hydrolyzed PAAC to palmitic acids and fluorogenic dye, coumarin. Release of coumarin was monitored by using fluorometer. NAAA hydrolyzed PAAC with an apparent Km of 20.05 μM and Vmax of 32.18 pmol/mg protein/min. This assay system can be used to develop inhibitors or activators of NAAA.
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KEYWORD
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Coumarin, Endocannabinoid, N-acylethanolamines, N-acylethanolamine-hydrolyzing acid amidase, Substrate
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