CLONING & EXPRESSION OF GROUP IV CYTOSOLIC CA2+ DEPENDENT PHOSPHOLIPASE

葡萄糖刺激胰岛产生磷脂 非酯化花生四烯酸的水解和积累 可能发挥胰岛素分泌的信号或效应作用。酶的种类 催化磷脂水解，胰岛细胞表达低 分子量分泌型磷脂酶A2(PLA2)和A组VI， 钙非依赖性磷脂酶A2(IPLA2)。先前的研究表明，小岛 还表达抗组IV的抗体识别的蛋白质， 胞浆内钙依赖的磷脂酶A2(CPLA2)。为了进一步研究 胰岛表达cPLA2的可能性，我们筛选了一个大鼠胰岛的基因 带有可识别cPLA2序列的探针的文库 全长cPLA2基因。大鼠胰岛cPLA2推导的氨基酸 序列与人和小鼠的cPLA2序列有96%的同源性。 CPLA2基因在COS-7细胞中的表达 钙依赖的磷脂酶A2活性和一种蛋白的诱导表达 被抗cPLA2抗体识别。比较 重组NT胰岛cPLA2和iPLA2活性在 转染的COS-7细胞表明iPLA2而不是cPLA2是 受到三磷酸腺苷的刺激。这两种活动对 花生四烯基三氟甲基酮的抑制作用，但iPLA2的作用更强 比起被卤代内酯自杀底物有效抑制 CPLA2。从纯化的胰岛细胞中提取RNA进行RT-PCR实验 从由荧光激活制备的富含？细胞的群体中 细胞分选结果显示cpla2基因在细胞中含量较高。 人口。免疫印迹分析表明，胰岛表达 CPLA2免疫反应蛋白和白介素1不影响 它的表情。因此，CPLA2是至少三类 具有不同性质的PLA2酶在细胞中表达。

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may play signaling or effector roles insulin secretion. Of enzymes that catalyze phospholipid hydrolysis, islet ?-cells express low molecular weight secretory phospholipases A2 (PLA2) and a Group VI, Ca2+-independent PLA2 (iPLA2). Previous studies indicate that islets also express a protein recognized by antibodies against a Group IV, cytosolic, Ca2+-dependent PLA2 (cPLA2). To further examine the possible expression of cPLA2 by islets, we screened a rat islet cDNA library with a probe that recognizes cPLA2 sequence and isolated a full-length cPLA2 cDNA. The rat islet cPLA2 deduced amino acid sequence is 96% identical to those of human and mouse cPLA2. Transfection of COS-7 cells with cPLA2 cDNA in an expression vector induced expression of Ca2+-dependent PLA2 activity and of a protein recognized by anti-cPLA2 antibody. Comparison of recombina nt islet cPLA2 and iPLA2 activities expressed in transfected COS-7 cells indicated that iPLA2 but not cPLA2 is stimulated by ATP. Both activities are similarly sensitive to inhibition by arachidonyltrifluoromethyl ketone, but iPLA2 is more effectively inhibited by a haloenol lactone suicide substrate than cPLA2. RT-PCR experiments with RNA from purified islet ?-cells and from an ?-cell-enriched population prepared by fluorescence-activated cell-sorting indicated that cPLA2 mRNA is more abundant in the ?-cell population. Immunoblotting analyses indicate that islets express cPLA2-immunoreactive protein and that interleukin-1 does not affect its expression. The cPLA2 is thus one of at least three classes of PLA2 enzymes with distinct properties expressed in ?-cells.