Arachidonoyl ethanolamide-[1,2-14C] as a substrate for anandamide amidase

Arachidonoyl ethanolamide-[1,2-14C] was prepared and evaluated as a substrate for anandamide amidase in a radioenzymatic assay that does not require a thin layer chromatography separation step. Using this substrate the release of ethanolamine-[1,2-14C] is linear for approximately thirty minutes. Anandamide amidase exhibits maximal activity between pH 8 and pH 9 with a steep decline in activity at pH values below 6 and above 10. Arachidonoyl ethanolamide-[1,2-14C] was used for the assay of anandamide amidase from 10 micrograms to 100 micrograms protein, from cow brain homogenate, in a 0.2 ml incubation mixture. When plotted as a rectangular hyperbola of the steady-state Michaelis-Menten equation, an approximate Km of 30 +/- 7 microM and a Vmax of 198 +/- 13 nmoles ethanolamine formed per hour per mg protein homogenate was obtained.