Enzymological and molecular biological studies on anandamide amidohydrolase

花生胺氨基水解酶是一种将花生胺(N-花生四烯酰乙醇胺)分解为花生四烯酸和乙醇胺的酰胺酶，N-花生四烯酰乙醇胺是大麻受体的内源性配体。阿南达胺在这种水解过程中失去了其拟大麻活性。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鼠肝脏的大量酶，并对该酶进行了高度纯化。用纯化的酶证明了苯乙酰胺酶解的可逆性，并测定了反应的平衡常数。我们还从猪脑中克隆了该酶的cDNA。当我们通过定点突变将该酶家族中高度保守的几个氨基酸残基改变为丙氨酸或其他氨基酸时，所有突变体的酶活性都很低或几乎检测不到。此外，我们还在人巨核母细胞系(CMK)中发现了脱氧核糖核酰胺的水解活性。催化该反应的酶不需要洗涤剂即可从膜上溶解，最适pH在5左右，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，对N-棕榈酰乙醇胺的催化活性高于对苯二胺的催化活性。根据这些发现，该酶被认为是一种不同于已知的苯乙酰胺氨基水解酶的同工酶。当我们对这种酶在大鼠各器官中的活性进行筛选时，发现肺的活性最高，其次是脾、胸腺、小肠等器官。该酶被吐温20有效地抑制，但被Triton X-100激活约7倍。经SDS-聚丙烯酰胺凝胶电泳法分析，高纯度的肺酶在31 kDa左右有一条主带。在各种N-酰基乙醇胺中，以N-棕榈酰乙醇胺的活性最高。

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.