Abstract
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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Amidohydrolases / analysis*
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Amidohydrolases / genetics
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Ammonia / metabolism
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Animals
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Arachidonic Acids / pharmacology
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Calcium Channel Blockers / pharmacology
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Cannabinoids / metabolism
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Drug Evaluation, Preclinical*
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Endocannabinoids
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Enzyme Inhibitors / pharmacology
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Glutamic Acid / metabolism
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Hydrolysis
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Kinetics
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Liver / enzymology
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Oleic Acids / metabolism
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Organophosphonates / pharmacology
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Phenylmethylsulfonyl Fluoride / pharmacology
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Polyunsaturated Alkamides
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Rats
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Rats, Inbred Strains
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Rats, Wistar
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Spectrophotometry / economics
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Spectrophotometry / methods*
Substances
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Arachidonic Acids
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Calcium Channel Blockers
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Cannabinoids
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Endocannabinoids
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Enzyme Inhibitors
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Oleic Acids
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Organophosphonates
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Polyunsaturated Alkamides
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methyl arachidonylfluorophosphonate
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Glutamic Acid
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Phenylmethylsulfonyl Fluoride
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Ammonia
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oleylamide
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Amidohydrolases
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fatty-acid amide hydrolase
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anandamide