A spectrophotometric assay for fatty acid amide hydrolase suitable for high-throughput screening

De Bank, Paul A.; Kendall, David A.; Alexander, Stephen P.H.

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 μM and 5.7 nmol/min/mg protein, 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. © 2005 Elsevier Inc. All rights reserved.

Citation

De Bank, P. A., Kendall, D. A., & Alexander, S. P. (2005). A spectrophotometric assay for fatty acid amide hydrolase suitable for high-throughput screening. Biochemical Pharmacology, 69(8), 1187-1193. https://doi.org/10.1016/j.bcp.2005.01.012

• Journal Article Type: Article
• Acceptance Date: Jan 24, 2005
• Online Publication Date: Mar 2, 2005
• Publication Date: Apr 15, 2005
• Deposit Date: Dec 3, 2022
• Journal: Biochemical Pharmacology
• Print ISSN: 0006-2952
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 69
• Issue: 8
• Pages: 1187-1193
• DOI: https://doi.org/10.1016/j.bcp.2005.01.012
• Public URL: https://nottingham-repository.worktribe.com/output/14322270
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0006295205000651?via%3Dihub