SYNTHESIS AND CHARACTERIZATION OF DIAZOMETHYLARACHIDONYL KETONE - AN IRREVERSIBLE INHIBITOR OF N-ARACHIDONYLETHANOLAMINE AMIDOHYDROLASE

N-Arachidonylethanolamine (AEA), a putative endogenous agonist of neur onal (CB1) cannabinoid receptors, is a substrate for N-arachidonyletha nolamine amidohydrolase (AEA amidohydrolase), a serine amidase present in cell membranes. Following a strategy that has been used to develop inhibitors that covalently bind to the active site of serine peptidas es, diazomethyl arachidonyl ketone (DAK) was synthesized and its effec ts on AEA amidohydrolase were determined. DAK inhibits the hydrolysis of AEA by rat brain membranes with an IC50 value of 0.5 mu M. At low c oncentrations, DAK reduces the V-max and increases the K-m of the enzy me for its substrate AEA, which suggests that it is both a competitive and noncompetitive inhibitor. At higher concentrations, DAK inhibitio n is completely noncompetitive. DAK inhibition of membrane-associated AEA amidohydrolase is irreversible because hydrolytic activity is not restored with extensive washing or dialysis of the membranes. Furtherm ore, DAK inhibition is not reversible by anion exchange chromatography of the subsequently solubilized enzyme. In contrast, DAK inhibition o f detergent-solubilized enzyme exhibits competitive kinetics and is re versible upon ion exchange chromatography. Exposure of C6 glioma cells to DAK results in concentration-related inhibition of AEA amidohydrol ase activity in cellular membranes with an IC50 value of 0.3 mu M. In summary, these studies demonstrate that DAK is an irreversible inhibit or of AEA amidohydrolase in its native membrane and provides a useful tool with which to study the role of AEA amidohydrolase in the termina tion of action of AEA.