FAAH activity was measured as the conversion of AEA labeled with [3H] in the ethanolamine portion of the molecule ([3H]AEA; Omeir et al., 1995) to [3H]ethanolamine preparations as reported previously (Hillard et al., 1995b). Membranes were incubated in a final volume of 0.5 ml of TME buffer (50 mM Tris-HCl, 3.0 mM MgCl2, and 1.0 mM EDTA, pH 7.4) containing 1.0 mg/ml fatty acid-free bovine serum albumin and 0.2 nM [3H]AEA. Isotherms were constructed using eight concentrations of AEA at concentrations between 10 nM and 10 μM. Incubations were carried out at 37°C and were stopped with the addition of 2 ml of chloroform/methanol (1:2). After standing at ambient temperature for 30 min, 0.67 ml of chloroform and 0.6 ml of water were added. Aqueous and organic phases were separated by centrifugation at 1,000 rpm for 10 min. The amount of [3H] in 1 ml each of the aqueous and organic phases was determined by liquid scintillation counting and the conversion of [3H]AEA to [3H]ethanolamine was calculated. The KI and Vmax values for this conversion were determined by fitting the data to a single site competition equation using Prism.
