Analysis of Fatty Acid Amide Hydrolase Activity in Plants

N
-Acylethanolamines (NAEs) are fatty acid derivatives amide-linked to ethanolamine. NAEs vary in chain lengths and numbers of double bonds and generally reflect the fatty acids found in membrane lipids in the tissues in which they reside. NAEs are present naturally in trace amounts and occur in a wide range of organisms including plants, animals, and microbes. Some NAE types are known to be involved in the endocannabinoid signaling system of vertebrates, and in plants they may play important regulatory roles in several physiological processes, such as root growth, seedling development, stress responses, and pathogen interactions. The biological effects of NAEs are terminated through their hydrolysis into the ethanolamine and free fatty acid by a membrane enzyme known as the fatty acid amide hydrolase (FAAH). Thus, FAAH represents an important target to better understand the function of these lipid mediators in numerous cellular processes. FAAH has been extensively characterized in mammalian and plant systems, and they share a conserved Ser-Ser-Lys catalytic mechanism. Here we describe procedures and experimental conditions to assay and characterize recombinant and endogenous FAAH enzymatic activity derived from plant tissues.