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.
Omeir
R L
RL
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794, USA.
Chin
S
S
Hong
Y
Y
Ahern
D G
DG
Deutsch
D G
DG
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D001921:Brain / Q000201:enzymology; D002250:Carbon Radioisotopes; D002417:Cattle; D063388:Endocannabinoids; D006863:Hydrogen-Ion Concentration; D053284:Polyunsaturated Alkamides; D051381:Rats; D013379:Substrate Specificity
D001095:Arachidonic Acids; D002250:Carbon Radioisotopes; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D000581:Amidohydrolases; C087226:arachidonoylethanolamide synthase; C078814:anandamide