Abstract
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.
Highlights
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 was shown that anandamide was rapidly degraded by an amidase activity which was found in the membrane fraction of cultured neuroblastoma and glioma cells and homogenates of rat tissues [9]
The pellet referred to as the microsomal fraction was chosen as a starting material for purification of the anandamide amidohydrolase
Summary
Anandamide and [1-14C]anandamide were chemically prepared from ethanolamine and nonradioactive or [1-14C]arachidonic acid, respectively, as described previously [15]. The resultant pellet (microsomal fraction, 300 mg of protein) was suspended in 42 ml of 50 mM Tris-HCl buffer (pH 8) containing 1% Triton X-100, kept for 12 h, and centrifuged at 105,000 ϫ g for 40 min. The supernatant was stored as the solubilized protein at Ϫ80 °C until use. The solubilized protein (6 –9 mg) was diluted in 20 ml of 20 mM citrate-sodium phosphate buffer (pH 6.0) containing 0.5 M ammonium sulfate and 0.05% Triton X-100 (solution A), passed through a 0.22-m membrane filter, and loaded onto a Tosoh Phenyl-5PW column (7.5 mm inside diameter ϫ 7.5 cm). Fractions with anandamide amidohydrolase activity of more than 0.2 nmol/min/100 l were pooled and stored at Ϫ80 °C. Protein concentration was determined by the method of Bradford [17] with bovine serum albumin as standard
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