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Enzymological and molecular biological studies on anandamide amidohydrolase

花生四烯酸乙醇胺酰胺水解酶的酶学和分子生物学研究

基本信息

批准号：
11670125
负责人：
UEDA Natsuo
金额：
$ 2.43万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670125/
关键词：
Cannabinoid Anandamide Arachidonic acid Amidase N-Acylethanolamine Marijuana Endocannabinoid Hydrolase

项目摘要

Anandamide amidohydrolase is an amidase hydrolyzing anandamide (N-arachidonoyl-ethanolamine), which was identified as an endogenous ligand of cannabinoid receptors, to arachidonic acid and ethanolamine. Anandamide loses its cannabimimetic activities by this hydrolysis. In this study we overexpressed large amounts of the enzyme of rat liver in a baculovirus-insect cell system, and highly purified the enzyme. With the purified enzyme we demonstrated the reversibility of the enzymatic hydrolysis of anandamide and determined the equilibrium constant of the reaction. We also cloned a cDNA of the enzyme from porcine brain. When we changed several amino acid residues of the enzyme, which are highly conserved in an amidase family including this enzyme, to alanine or other amino acids by site-directed mutagenesis, the enzymatic activities of all the mutants were very low or hardly detectable. Furthermore, we discovered an anandamide hydrolyzing activity in a human megakaryoblastic cell line (CMK). The enzyme catalyzing this reaction could be solubilized from the membrane without detergent, had an optimal pH around 5, showed low sensitivity to serine hydrolase inhibitors, was activated by dithiothreitol, and was more active with N-palmitoylethanolamine than with anandamide. Based on these findings the enzyme was thought to be an isozyme distinct from the known anandamide amidohydrolase. When we screened for such an enzyme activity in various rat organs, the highest activity was detected in lung, followed by spleen, thymus, small intestine and other organs. The enzyme was potently inhibited by Tween 20, but was activated around seven fold by Triton X-100. As analyzed by SDS-polyacrylamide gel electrophoresis, the highly purified enzyme of lung showed a major band around 31 kDa. The purified enzyme was the most active with N-palmitoylethanolamine among various N-acylethanolamines.

Anandamide 酰胺水解酶是一种酰胺酶，可将 Anandamide（N-花生四烯酰乙醇胺）水解为花生四烯酸和乙醇胺，该酶被鉴定为大麻素受体的内源配体。通过这种水解，花生四烯乙醇胺失去了其大麻模拟活性。在这项研究中，我们在杆状病毒-昆虫细胞系统中过量表达了大量大鼠肝脏的酶，并高度纯化了该酶。使用纯化的酶，我们证明了 anandamide 酶水解的可逆性，并确定了反应的平衡常数。我们还从猪脑中克隆了该酶的 cDNA。当我们通过定点诱变将酶的几个氨基酸残基（在包括该酶在内的酰胺酶家族中高度保守）改变为丙氨酸或其他氨基酸时，所有突变体的酶活性都非常低或几乎检测不到。此外，我们在人巨核细胞系 (CMK) 中发现了 anandamide 水解活性。催化该反应的酶无需洗涤剂即可从膜上溶解，最佳pH值约为5，对丝氨酸水解酶抑制剂的敏感性较低，可被二硫苏糖醇激活，并且与N-棕榈酰乙醇胺相比，与anandamide相比，其活性更高。基于这些发现，该酶被认为是与已知的花生四烯酸乙醇胺酰胺水解酶不同的同工酶。当我们在大鼠各个器官中筛选这种酶的活性时，在肺中检测到的活性最高，其次是脾脏、胸腺、小肠和其他器官。该酶被 Tween 20 有效抑制，但被 Triton X-100 激活约七倍。通过SDS-聚丙烯酰胺凝胶电泳分析，高度纯化的肺酶在31 kDa附近显示出主要条带。纯化后的酶在各种N-酰基乙醇胺中对N-棕榈酰乙醇胺的活性最强。