Partial purification and characterization of the porcine brain enzyme hydrolyzing and synthesizing anandamide.
Anandamide (arachidonylethanolamide) is known as an endogenous agonist for cannabinoid receptors. An amidohydrolase, which hydrolyzed anandamide, was solubilized from the microsomal fraction of porcine brain with 1% Triton X-100. The enzyme was partially purified by Phenyl-5PW hydrophobic chromatography to a specific activity of approximately 0.37 mumol/min/mg of protein at 37 degrees C. As assayed with 14C-labeled substrates, the apparent Km value for anandamide was 60 microM, and anandamide was more active than ethanolamides of linoleic, oleic, and palmitic acids. Ceramidase and protease activities were not detected in our enzyme preparation. The purified enzyme also synthesized anandamide from free arachidonic acid in the presence of a high concentration of ethanolamine with a specific activity of about 0.16 mumol/min/mg of protein at 37 degrees C. On the basis of cochromatographies, pH dependence, heat inactivation, and effects of inhibitors such as arachidonyl trifluoromethyl ketone, p-chloromercuribenzoic acid, diisopropyl fluorophosphate, and phenylmethylsulfonyl fluoride, it was suggested that the anandamide amidohydrolase and synthase activities were attributable to a single enzyme protein.
Ueda
N
N
Department of Biochemistry, Tokushima University, School of Medicine, Japan.
Kurahashi
Y
Y
Yamamoto
S
S
Tokunaga
T
T
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001095:Arachidonic Acids / Q000096:biosynthesis / Q000378:metabolism*; D001921:Brain / Q000201:enzymology*; D002186:Cannabinoids / Q000096:biosynthesis / Q000378:metabolism; D063388:Endocannabinoids; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D013379:Substrate Specificity; D013552:Swine; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D002186:Cannabinoids; D063388:Endocannabinoids; D009842:Oligopeptides; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C087226:arachidonoylethanolamide synthase; C078814:anandamide