oleylamide and methyl-arachidonylfluorophosphonate

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.

Topics: Amidohydrolases; Ammonia; Animals; Arachidonic Acids; Calcium Channel Blockers; Cannabinoids; Drug Evaluation, Preclinical; Endocannabinoids; Enzyme Inhibitors; Glutamic Acid; Hydrolysis; Kinetics; Liver; Oleic Acids; Organophosphonates; Phenylmethylsulfonyl Fluoride; Polyunsaturated Alkamides; Rats; Rats, Inbred Strains; Rats, Wistar; Spectrophotometry