Molecular characterization of a phospholipase D generating anandamide and its congeners
Molecular characterization of a phospholipase D generating anandamide and its congeners
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DOI：
10.1074/jbc.m306642200
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发表时间：
2004-02-13
影响因子：
4.8
通讯作者：
Ueda, N
中科院分区：
文献类型：
--
作者：
Okamoto, Y;Morishita, J;Ueda, N
花生四烯酰胺（N-花生四烯酰乙醇胺）是已知的大麻素和香草素受体的内源性配体。其同系物（统称为N-酰基乙醇胺）也表现出多种生物活性。这些化合物主要由动物组织中磷脂酶D型的磷酸二酯酶从其相应的N-酰基-磷脂酰乙醇胺形成。我们从大鼠心脏中纯化了该酶，并利用其内部肽序列从小鼠、大鼠和人中克隆了其互补DNA。推导的氨基酸序列由393-396个残基组成，并表明该酶与已知的磷脂酶D酶没有同源性，但被分类为β-内酰胺酶折叠的锌金属水解酶家族的成员。由于在COS-7细胞中过表达，重组酶以相当的速率从其相应的N-酰基-磷脂酰乙醇胺产生花生四烯酸和其他N-酰基乙醇胺。相反，该酶与磷脂酰胆碱和磷脂酰乙醇胺无活性。酶活性和信使RNA和蛋白质水平的测定表明，其广泛分布在小鼠器官中，在脑、肾和睾丸中含量较高。这些结果证实，一个特定的磷脂酶D是负责产生N-酰基乙醇胺，包括花生四烯酸，强烈表明这类脂质分子的生理重要性。
Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors. Its congeners (collectively referred to as N-acylethanolamines) also show a variety of biological activities. These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues. We purified the enzyme from rat heart, and by the use of the sequences of its internal peptides cloned its complementary DNAs from mouse, rat, and human. The deduced amino acid sequences were composed of 393-396 residues, and showed that the enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the beta-lactamase fold. As was overexpressed in COS-7 cells, the recombinant enzyme generated anandamide and other N-acylethanolamines from their corresponding N-acyl-phosphatidylethanolamines at comparable rates. In contrast, the enzyme was inactive with phosphatidylcholine and phosphatidylethanolamine. Assays of the enzyme activity and the messenger RNA and protein levels revealed its wide distribution in murine organs with higher contents in the brain, kidney, and testis. These results confirm that a specific phospholipase D is responsible for the generation of N-acylethanolamines including anandamide, strongly suggesting the physiological importance of lipid molecules of this class.