A radiochromatographic method has been set up in order to determine fatty acid amide hydrolase (FAAH) activity, based on reversed-phase high- performance liquid chromatography and on-line scintillation counting. The reaction products were separated using a C18 column eluted with methanol- water-acetic acid and quantitated with an external standard. Baseline separation of the acid product from the substrate was completed in less than 4 min, with a detection limit of 2.5 fmol arachidonic acid at a signal to noise ratio of 4:1. The method enabled to determine the kinetic constants (i.e., apparent K(m) of 2.0 ± 0.2 μM and V(max) of 800 ± 75 pmol · min- 1 · mg protein-1 toward anandamide) and the substrate specificity of human brain FAAH, as well as the extent of enzyme inhibition by some anandamide congeners. The femtomole sensitivity and the accuracy of the method allow detection and characterization of the activity of FAAH in very minute tissue samples or in samples where the enzymatic activity is very low.
ASJC Scopus subject areas
- Molecular Biology