Studies on an enzyme hydrolyzing anandamide, an endogenous ligand for cannabinoid receptors

大麻素是大麻中含有的精神活性成分，其特异性受体在脑和其他器官中表达。花生四烯酸乙醇酰胺（arachidonoyethanolamide）是大麻素受体的内源性配体，在水解成花生四烯酸和乙醇胺后失去其拟大麻生物活性。为了阐明花生四烯酸酰胺水解酶催化该反应的催化特性，用大鼠肝酶的cDNA转染COS-7细胞，并表达重组酶。该酶不仅具有花生四烯酸水解酶活性，而且具有花生四烯酸与乙醇胺缩合的花生四烯酸合成酶活性。在对照COS-7细胞中，这两种反应均未发生。这些结果证明了花生四烯酸酰胺水解的可逆性。然而，由于乙醇胺的高Km，该酶在生理条件下似乎充当花生四烯酸水解酶而不是花生四烯酸合酶。除了酰胺酶活性外，该酶还表现出对花生四烯酸甲酯的酯酶活性，因此，我检查了该酶是否水解大麻素受体的另一种配体2-花生四烯酸酰甘油（2-AG）的酯键。结果表明，2-AG的水解速度比花生四烯酸的水解速度快4倍左右，Km值低至6 μ M，最适pH为10，而对照细胞几乎检测不到这种水解反应。两种化合物同时抑制花生四烯酸和2-AG的水解，花生四烯酸抑制2-AG的水解呈剂量依赖性。这些结果表明，花生四烯酸和2-AG可以被相同的酶降解。

Cannabinoids are psychoactive components contained in Cannabis sativa L.and their specific receptors are expressed in brain and other organs. Anandamide (arachidonoylethanolamide) was found as an endogenous ligand for the cannabinoid receptors, and reported to lose its cannabimimetic biological activities by the hydrolysis to arachidonic acid and ethanolamine. In order to elucidate the catalytic properties of anandamide amidohydrolase catalyzing this reaction, COS-7 cells were transfected with cDNA for the rat liver enzyme, and the recombinant enzyme was expressed. The enzyme showed not only the anandamide hydrolase activity but also its reverse reaction, namely, the anandamide synthase activity by the condensation of arachidonic acid with ethanolamine. Both the reactions did not occur in the control COS-7 cells. These results demonstrated the reversibility of the anandamide hydrolysis. However, due to a high Km for ethanolamine, the enzyme seemed to act as an anandamide hydrolase rather than an anandamide synthase under the physiological conditions. In addition to the amidase activity, the enzyme showed an esterase activity for methyl arachidonate.Therefore, I examined whether or not the enzyme hydrolyzes the ester bond of 2-arachidonoylglycerol (2-AG), another ligand for the cannabinoid receptors. The results showed that the hydrolysis of 2-AG proceeded about 4-fold faster than the anandamide hydrolysis with a Km value as low as 6muM and an optimal pH of IO.This hydrolytic reaction was hardly detected with the control cells. Two compounds inhibited the hydrolysis of both anandamide and 2-AG in parallel, and anandamide inhibited the hydrolysis of 2-AG dose-dependently. These results indicate that anandamide and 2-AG can be degraded by the same enzyme.