TY - JOUR
T1 - A spectrophotometric assay for fatty acid amide hydrolase suitable for high-throughput screening
AU - De Bank, P A
AU - Kendall, D A
AU - Alexander, S P H
PY - 2005
Y1 - 2005
N2 - 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 K-m and Y-max values of 104 mu M 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
AB - 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 K-m and Y-max values of 104 mu M 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
UR - https://www.scopus.com/pages/publications/15744389868
U2 - 10.1016/j.bcp.2005.01.012
DO - 10.1016/j.bcp.2005.01.012
M3 - Article
SN - 0006-2952
VL - 69
SP - 1187
EP - 1193
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
ER -