Chen Y, Dennis E A
Department of Chemistry and Biochemistry, School of Medicine and Revelle College, University of California, San Diego, La Jolla, CA 92093-0601, USA.
Biochim Biophys Acta. 1998 Oct 2;1394(1):57-64. doi: 10.1016/s0005-2760(98)00098-8.
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.
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。我们报告了GV-PLA2的过表达及特性。通过逆转录聚合酶链反应（RT-PCR）从人心脏多聚腺苷酸加尾mRNA（polyA+ mRNA）合成GV-PLA2互补DNA（cDNA），并构建了包含GV-PLA2的表达载体。在大肠杆菌细胞中表达后，使用镍亲和层析一步将该蛋白溶解并纯化至同质状态。纯化后的GV-PLA2蛋白折叠形成活性酶。重组GV-PLA2的酶活性绝对依赖于钙离子。以超声处理的囊泡为底物时，该酶在Tris-HCl缓冲液中的最适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，在磷脂混合囊泡中，PC和PE的水解情况相当。对于具有PC头部基团和超声处理囊泡的情况，GV-PLA2对脂肪酸的偏好顺序为亚油酰基>棕榈酰基>花生四烯酰基。3-(3-乙酰胺-1-苄基-2-乙基吲哚-5-氧基)丙烷膦酸（LY311727）是一种有效的人IIA组磷脂酶A2抑制剂，它对GV-PLA2有强烈抑制作用，半数抑制浓度（IC50）值约为36 nM，这与其对IIA组磷脂酶A2的抑制作用相当。
