Unknown

FAAH activity was measured by following the production of ammonia generated from the hydrolysis of oleamide by FAAH. GDH catalyzes the condensation of ammonia and R-ketoglutarate to glutamate with a concomitant conversion of NADH to nicotinamide adenine dinucleotide, oxidized form (NAD+), which is spectrophotometrically measured at 340 nm. To determine IC50, the reactions were carried out in 96-well clear polystyrene plates in a total volume of 200 uL. To a reaction mixture (140 uL) containing NaPi, pH 7.4, NADH, R-ketoglutarate, ADP, EDTA, and GDH to final concentrations of 50 mM, 150 uM, 3 mM, 2 mM, 1 mM, and 7.5 unit/mL, respectively, was added 20 uL of PF-750 dissolved in 50% DMSO (20 uL of 50% DMSO for controls). After the resulting mixture was mixed in a plate vortex, 20 uL of hFAAH (370 ng) in 20 mM NaPi, pH 7.8/1% Triton X-100, was added and mixed. After this mixture was preincubated at RT for the indicated period of time (5-60 min), the reaction was initiated by addition of 20 uL of 500 uM oleamide dissolved in 25% DMSO and 75% EtOH.The reactions were incubated at 30 °C, and the absorbance at 340 nm was collected over a period of 30 min. IC50 for PF-750 was time-dependend, and varied from 0.595 uM at 5 min pre-incubation to 0.0162 uM at 60 min pre-incubation