Expression and characterization of human group V phospholipase A2

Yijun Chen, Edward A. Dennis
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引用次数: 81

Abstract

Group V phospholipase A2 (GV-PLA2) has been shown to be involved in signal transduction and inflammatory processes in cellular studies, but the physical and biochemical properties of this important enzyme have been unclear. We report the over-expression and characterization of GV-PLA2. The GV-PLA2 cDNA was synthesized from human heart polyA+ mRNA by RT-PCR, and an expression construct containing the GV-PLA2 was established. After expression in Escherichia coli cells, the protein was solubilized and purified to homogeneity in a single step using nickel affinity chromatography. The purified GV-PLA2 protein was folded to form active enzyme. The recombinant GV-PLA2 has an absolute requirement for Ca2+ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with sonicated vesicles as substrate. GV-PLA2 preferentially hydrolyzes phosphatidylethanolamine (PE) vesicles compared to phosphatidylcholine (PC) vesicles. However, hydrolysis of PC and PE is equivalent in mixed vesicles of the phospholipids. The fatty acid preference of GV-PLA2 is linoleoyl>palmitoyl>arachidonyl with a PC head group and sonicated vesicles. 3-(3-Actamide-1-benzyl-2-ethylindolyl-5-oxy)propane phosphonic acid (LY311727), a potent inhibitor of human group IIA PLA2, strongly inhibits GV-PLA2 with an IC50 value of about 36 nM which is comparable to its inhibition of group IIA PLA2.

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人V组磷脂酶A2的表达与特性研究
在细胞研究中,V组磷脂酶A2 (GV-PLA2)已被证明参与信号转导和炎症过程,但这种重要酶的物理和生化特性尚不清楚。我们报道了GV-PLA2的过表达和特征。采用RT-PCR方法从人心脏polyA+ mRNA合成GV-PLA2 cDNA,并构建了含GV-PLA2的表达构建体。在大肠杆菌细胞中表达后,用镍亲和层析法将蛋白一次性溶解纯化至均匀。将纯化的GV-PLA2蛋白折叠形成活性酶。重组GV-PLA2酶活性绝对需要Ca2+。该酶在以超声囊泡为底物的Tris-HCl缓冲液中最适pH为8.5。与磷脂酰胆碱(PC)囊泡相比,GV-PLA2优先水解磷脂酰乙醇胺(PE)囊泡。然而,PC和PE的水解在磷脂混合囊泡中是等效的。GV-PLA2的脂肪酸偏好是具有PC头基团和超声囊泡的亚油基棕榈基花生四烯基。3-(3- actamide -1-苄基-2-乙基lindolyl-5-氧基)丙烷膦酸(LY311727)是人IIA PLA2组的有效抑制剂,对GV-PLA2具有较强的抑制作用,IC50值约为36 nM,与对IIA PLA2组的抑制作用相当。
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