Desarnaud F, Cadas H, Piomelli D
Unité de Neurobiologie et Pharmacologie, Centre P. Broca de l'INSERM, Paris, France.
J Biol Chem. 1995 Mar 17;270(11):6030-5. doi: 10.1074/jbc.270.11.6030.
An amidohydrolase activity present in rat brain microsomes catalyzes the hydrolysis of N-arachidonoyl-[3H]ethanolamine ([3H]anandamide), an endogenous cannabimimetic substance, forming [3H]ethanolamine and arachidonic acid. Amidohydrolase activity is maximal at pH 6 and 8, is independent of divalent cations, has an apparent Km for [3H]anandamide of 12.7 +/- 1.8 microM, and has a Vmax of 5630 +/- 200 pmol/min/mg of protein. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, and p-bromophenacyl bromide, a histidine-alkylating reagent, inhibit the activity, whereas N-ethylmaleimide and various nonselective peptidase inhibitors (EDTA, o-phenanthroline, bacitracin) have no effect. Brain amidohydrolase activity exhibits high substrate specificity for [3H]anandamide; N-gamma-linolenoyl-, N-homo-gamma-linolenoyl-, and N-11,14-eicosadienoyl- are hydrolyzed at markedly slower rates. Moreover, N-11-eicosaenoyl- and N-palmitoyl-[3H]ethanolamine are not hydrolyzed. [3H]Anandamide hydrolysis is inhibited competitively by nonradioactive anandamide and by other N-acylethanolamines with the following rank order of potency: anandamide > N-linoleoyl- = N-cis-linolenoyl- = N-gamma-linolenoyl- = N- homo-gamma-linolenoyl- > N-11,14-eicosadienoyl- > N-oleoyl- > N- docosahexaenoyl- > N-docosatetraenoyl > N-linoelaidoyl- > N-eicosaenoyl- > N- palmitoyl > or = N-elaidoyl- = N-eicosanoyl-ethanolamine = no effect. Amidohydrolase activity is high in liver and brain and low in heart, kidney, intestine, stomach, lung, spleen, and skeletal muscle. Within the central nervous system, highest activity is found in globus pallidus and hippocampus, two regions rich in cannabinoid receptors, and lowest activity is found in brainstem and medulla, where cannabinoid receptors are sparse. The results, showing that brain amidohydrolase activity is selective for anandamide and enriched in areas of the central nervous system with high density of cannabinoid receptors, suggest that this activity may participate in the inactivation of anandamide at its sites of action.
大鼠脑微粒体中存在的一种酰胺水解酶活性可催化内源性大麻素样物质N-花生四烯酰基-[3H]乙醇胺（[3H]花生四烯酸乙醇胺）水解，生成[3H]乙醇胺和花生四烯酸。酰胺水解酶活性在pH 6和8时最高，不依赖于二价阳离子，对[3H]花生四烯酸乙醇胺的表观Km为12.7±1.8 μM，Vmax为5630±200 pmol/分钟/毫克蛋白质。丝氨酸蛋白酶抑制剂苯甲基磺酰氟和组氨酸烷基化试剂对溴苯甲酰溴可抑制该活性，而N-乙基马来酰亚胺和各种非选择性肽酶抑制剂（乙二胺四乙酸、邻菲罗啉、杆菌肽）则无作用。脑酰胺水解酶活性对[3H]花生四烯酸乙醇胺表现出高底物特异性；N-γ-亚麻酸基-、N-同型-γ-亚麻酸基-和N-11,14-二十碳二烯酰基-的水解速度明显较慢。此外，N-11-二十碳烯酰基-和N-棕榈酰基-[3H]乙醇胺不被水解。非放射性花生四烯酸乙醇胺和其他N-酰基乙醇胺竞争性抑制[3H]花生四烯酸乙醇胺水解，其效力顺序如下：花生四烯酸乙醇胺＞N-亚油酰基- = N-顺式-亚麻酸基- = N-γ-亚麻酸基- = N-同型-γ-亚麻酸基-＞N-11,14-二十碳二烯酰基-＞N-油酰基-＞N-二十二碳六烯酰基-＞N-二十二碳四烯酰基＞N-反式-亚油酰基-＞N-二十碳烯酰基-＞N-棕榈酰基＞或 = N-反油酰基- = N-二十碳酰基-乙醇胺 = 无作用。肝脏和脑中的酰胺水解酶活性高，而心脏、肾脏、肠道、胃、肺、脾脏和骨骼肌中的活性低。在中枢神经系统中，苍白球和海马体中的活性最高，这两个区域富含大麻素受体，而脑干和延髓中的活性最低，大麻素受体在这些部位分布稀疏。结果表明，脑酰胺水解酶活性对花生四烯酸乙醇胺具有选择性，且在大麻素受体高密度的中枢神经系统区域富集，提示该活性可能参与花生四烯酸乙醇胺在其作用部位的失活过程。
