A fluorescence displacement assay for the measurement of arachidonoyl ethanolamide (anandamide) and oleoyl amide (octadecenoamide) hydrolysis.
We describe a simple fluorescence displacement assay to measure hydrolysis of arachidonoyl ethanolamide and oleoyl amide, two important pharmacological compounds. Hydrolysis at the amide linkage of these ligands releases a fatty acid as one of the products. The displacement of a fluorescent fatty acid analogue from rat liver fatty acid-binding protein by the released fatty acid can thus be measured as a decrease in fluorescence. This process is time- and concentration-dependent and shows hyperbolic enzyme kinetics. Electrospray ionisation mass spectrometry was used to validate the assay.
Thumser
A E
AE
Department of Biochemistry, University of Southampton, United Kingdom.
Voysey
J
J
Wilton
D C
DC
D000577:Amides / Q000378:metabolism; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D002186:Cannabinoids / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D063388:Endocannabinoids; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005453:Fluorescence; D006868:Hydrolysis; D018996:Myelin P2 Protein / Q000378:metabolism; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009829:Oleic Acids / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D051381:Rats; D012890:Sleep / Q000187:drug effects
D000577:Amides; D001095:Arachidonic Acids; D002186:Cannabinoids; D002352:Carrier Proteins; D063388:Endocannabinoids; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009829:Oleic Acids; D053284:Polyunsaturated Alkamides; C078814:anandamide