A simple stopped assay for fatty acid amide hydrolase avoiding the use of a chloroform extraction phase.
A stopped assay for fatty acid amide hydrolase (FAAH) has been developed, whereby the enzyme reaction product ([(3)H]ethanolamine) was separated from substrate (anandamide [ethanolamine-1-(3)H]), by differential adsorption to charcoal. The assay gave a better extraction efficiency when acidic rather than alkaline charcoal solutions were used to stop the reaction, and a very good ratio of sample/blank was also seen. The acidic charcoal assay gave the expected sensitivities to compounds known to inhibit FAAH (palmitoyltrifluoromethyl ketone, arvanil, AM404 and indomethacin). It is concluded that the acidic charcoal extraction method provides a robust and simple stopped assay for FAAH without the need to use potentially hazardous solvents like chloroform.
Boldrup
Linda
L
Department of Pharmacology and Clinical Neuroscience, Umeå University, SE901 87 Umeå, Sweden.
Wilson
Sandy J
SJ
Barbier
Ann J
AJ
Fowler
Christopher J
CJ
D000327:Adsorption; D000581:Amidohydrolases / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry; D001703:Biophysics / Q000379:methods*; D001921:Brain / Q000378:metabolism; D002606:Charcoal / Q000737:chemistry*; D002725:Chloroform / Q000737:chemistry / Q000494:pharmacology*; D063388:Endocannabinoids; D019856:Ethanolamine / Q000737:chemistry; D053284:Polyunsaturated Alkamides; D051381:Rats; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D002606:Charcoal; D002725:Chloroform; D063388:Endocannabinoids; D019856:Ethanolamine; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide