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 mol/min/mg of protein at 37°C. As assayed with 14 C-labeled substrates, the apparent K m value for anandamide was 60 M, 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 mol/min/mg of protein at 37°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.An endogenous agonist for cannabinoid receptor was isolated from porcine brain, and this compound referred to as anandamide was identified to be arachidonylethanolamide (1). It inhibited the specific binding of radiolabeled ligands to cannabinoid receptors, reduced cAMP production, and caused the inhibition of N-type calcium currents and calcium channel antagonist binding (1-5). Anandamide also inhibited electrically evoked contraction of vas deferens isolated from mice (1) and mimicked in vivo effects of cannabinoids such as antinociception, hypothermia, hypoactivity, and catalepsy in mice (6 -8).It was shown that anandamide was rapidly degraded by an amidase (amidohydrolase) activity which was found in the membrane fraction of cultured neuroblastoma and glioma cells and homogenates of rat tissues (9). In fact, the addition of PMSF, 1 a serine protease inhibitor (9), increased an apparent affinity of anandamide for cannabinoid receptors, probably due to protecting the compound from hydrolysis (10, 11). Recently, some properties of the enzyme were reported with a microsomal preparation of rat brain (12). On the other hand, the synthesis of anandamide from free arachidonic acid and ethanolamine was shown with rat (9), bovine (13), and rabbit (14) brain and was reported to be independent of ATP and coenzyme A (14). However, the enzyme(s) hydrolyzing and synthesizing anandamide has not yet been purified and well characterized, and it is still unknown whether the two enzyme activities are attributed to a single enzyme protein or two enzymes. Enzyme Preparation-Porcine brain was obtained at a local slaughterhouse. The brain (approximately 100 g) was homogenized in 9 times the volume (v/w) of ice-cold 20 mM Tris-HCl (pH 8) containing 0.32 M sucrose with a Potter-Elvehjem homogenizer. The following procedures were performed at 4°C. The homogenate ...
