ARACHIDONOYL ETHANOLAMIDE-[1,2-C-14] AS A SUBSTRATE FOR ANANDAMIDE AMIDASE

Arachidonoyl ethanolamide-[1,2-C-14] was prepared and evaluated as a s ubstrate for anandamide amidase in a radioenzymatic assay that does no t require a thin layer chromatography separation step. Using this subs trate the release of ethanolamine-[1,2-C-14] is linear for approximate ly thirty minutes. Anandamide amidase exhibits maximal activity betwee n pH 8 and pH 9 with a steep decline in activity at pH values below 6 and above 10. Arachidonoyl ethanolamide-[1,2- C-14] was used for the a ssay of anandamide amidase from 10 mu g to 100 mu g protein, from cow brain homogenate, in a 0.2 ml incubation mixture. When plotted as a re ctangular hyperbola of the steady-state Michaelis-Menten equation, an approximate K-m of 30 +/- 7 mu M and a V-max of 198 +/- 13 nmoles etha nolamine formed per hour per mg protein homogenate was obtained.