A spectrophotometric assay for fatty acid amide hydrolase suitable for high-throughput screening.
Signalling via the endocannabinoids anandamide and 2-arachidonylglycerol appears to be terminated largely through the action of the enzyme fatty acid amide hydrolase (FAAH). In this report, we describe a simple spectrophotometric assay to detect FAAH activity in vitro using the ability of the enzyme to hydrolyze oleamide and measuring the resultant production of ammonia with a NADH/NAD+-coupled enzyme reaction. This dual-enzyme assay was used to determine Km and Vmax values of 104 microM and 5.7 nmol/min/mgprotein, respectively, for rat liver FAAH-catalyzed oleamide hydrolysis. Inhibitor potency was determined with the resultant rank order of methyl arachidonyl fluorophosphonate>phenylmethylsulphonyl fluoride>anandamide. This assay system was also adapted for use in microtiter plates and its ability to detect a known inhibitor of FAAH demonstrated, highlighting its potential for use in high-throughput screening.
De Bank
Paul A
PA
School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK. paul.debank@nottingham.ac.uk
Kendall
David A
DA
Alexander
Stephen P H
SP
D000581:Amidohydrolases / Q000032:analysis* / Q000235:genetics; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002186:Cannabinoids / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009829:Oleic Acids / Q000378:metabolism; D063065:Organophosphonates / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D053284:Polyunsaturated Alkamides; D051381:Rats; D011919:Rats, Inbred Strains; D017208:Rats, Wistar; D013053:Spectrophotometry / Q000191:economics / Q000379:methods*; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D000641:Ammonia; D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D002186:Cannabinoids; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D018698:Glutamic Acid; D009829:Oleic Acids; D063065:Organophosphonates; D010664:Phenylmethylsulfonyl Fluoride; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C104810:methyl arachidonylfluorophosphonate; C029407:oleylamide; C078814:anandamide