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

大麻素类化合物是大麻中的精神活性成分，其特异性受体在大脑和其他器官中表达。花生胺(花生四烯基乙醇胺)被发现是大麻素受体的内源性配体，据报道，由于花生四烯酸和乙醇胺的水解而失去了拟大麻的生物活性。为了阐明氨基水解酶催化这一反应的性质，将大鼠肝酶基因导入COS-7细胞，并表达重组酶。该酶不仅显示了花生胺水解酶的活性，而且还表现出其反向反应，即花生四烯酸与乙醇胺缩合而产生的花生胺合成酶活性。这两种反应在对照组COS-7细胞中均未发生。这些结果证明了苯乙酰胺水解的可逆性。然而，由于乙醇胺的Km很高，在生理条件下，该酶似乎是一种苯乙酰胺水解酶，而不是一种二乙醇胺合成酶。除了酰胺酶的活性外，该酶还表现出对花生四烯酸甲酯的酯酶活性。因此，我检测了该酶是否能水解大麻受体的另一种配体2-花生四烯基甘油(2-AG)的酯键。结果表明，2-氨基葡聚糖的Km值低至6µM，最适pH为1，2-AG的水解率约为ANANDAME的4倍，而对照细胞几乎检测不到该反应。两个化合物同时抑制ANANDAME和2-AG的水解率，且ANANDAME呈剂量依赖关系抑制2-AG的水解率。这些结果表明，花生胺和2-氨基酚可以被同一种酶降解。

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.