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Functional analysis of a novel enzyme of the phospholipase D type involved in the endocannabinoid

参与内源性大麻素的新型磷脂酶 D 型酶的功能分析

基本信息

批准号：
17590251
负责人：
UEDA Natsuo
金额：
$ 2.24万
依托单位：
Kagawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Anandamide (N-arachidonoylethanolamine), which was found as an endogenous ligand for cannabinoid receptors (an endocannabinoid), is formed from membrane glycerophospholipids by two-step enzyme reactions in animal tissues. However, the responsible enzymes remained poorly characterized, and it has been difficult to study the anandamide biosynthesis by molecular biological approaches. Recently we succeeded for the first time in cDNA cloning and functional expression of a novel mammalian enzyme of the phospholipase D type (NAPE-PLD) that generates anandamide and other N-acylethanolamines from their corresponding N-acylphosphatidylethanolamines (NAPEs) (Okamoto et al. J. Biol. Chem. 279, 5298-305, 2004). In the present study, we principally characterized recombinant rat NAPE-PLD.The recombinant NAPE-PLD was expressed in Escherichia coli as a GST-fusion protein together with molecular chaperone. The enzyme was then solubilized with CHAPS, followed by purification to apparent homogeneity by glutathione affinity chromatography and hydroxyapatite chromatography. The purified enzyme was highly active with NAPEs, and did not discriminate various N-acyl species with C_4-C_<20>. In contrast, the enzyme was almost inactive with major membrane glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol). These results suggested the ability of NAPE-PLD to specifically degrade different NAPEs without damaging other membrane phospholipids. The purified enzyme was remarkably activated in a dose-dependent manner by millimolar concentrations of Mg^<2+> as well as Ca^<2+>. Atomic absorption spectrometry exhibited the presence of catalytically important zinc in NAPE-PLD. In addition, site-directed mutagenesis studies revealed that Asp-147, His-185, His-187, Asp-189, His-190, His-253, Asp-284, and His-321 of NAPE-PLD, that are highly conserved within the metallo-□-lactamase family, play crucial roles in the catalytic activity.

大麻素受体（一种内源性大麻素）的内源性配体，在动物组织中通过两步酶反应由膜甘油磷脂形成。然而，相关酶的特性仍然很差，并且很难通过分子生物学方法来研究花生四烯酸的生物合成。最近，我们首次成功地进行了磷脂酶D型（NAPE-PLD）的新哺乳动物酶的cDNA克隆和功能表达，该酶从其相应的N-酰基磷脂酰乙醇胺（NAPE）产生花生四烯酸和其它N-酰基乙醇胺（Okamoto et al.J.Biol.Chem.279，5298-305，2004）。在本研究中，我们主要研究了重组大鼠NAPE PLD，重组NAPE PLD在大肠杆菌中以GST融合蛋白的形式与分子伴侣一起表达。然后用CHAPS溶解酶，然后通过谷胱甘肽亲和层析和羟基磷灰石层析纯化至表观均一性。纯化的酶对NAPEs有很高的活性，对C_4-C_4的各种N-酰基没有区分能力<20>。相反，酶几乎是无活性的主要膜甘油磷脂（磷脂酰胆碱，磷脂酰乙醇胺，磷脂酰丝氨酸，磷脂酰肌醇）。这些结果表明NAPE-PLD特异性降解不同NAPE而不损伤其他膜磷脂的能力。纯化的酶被毫克摩尔浓度的Mg^2+和Ca^2+以剂量依赖的方式显著激活。原子吸收光谱法显示NAPE PLD中存在催化重要的锌。此外，定点突变研究表明，在金属-□-内酰胺酶家族中高度保守的NAPE-PLD的Asp-147、His-185、His-187、Asp-189、His-190、His-253、Asp-284和His-321在催化活性中起关键作用。