CLONE IV CYTOSOLIC CA2+ DEPENDENT PHOSPHOLIPASE A2 FROM RAT PANCREATIC ISLETS

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 由同位素稀释质谱学证明，这可能会起到 信号或效应器起着胰岛素分泌的作用。一系列的酶 催化磷脂水解，胰岛b细胞表达低分子 重量分泌型磷脂酶A2(PLA2)和第VI组， 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 用抗cPLA2抗体识别。重组胰岛的比较 CPLA2和iPLA2在COS-7细胞中的表达 提示iPLA2受ATP刺激，而cPLA2不受其刺激。两者都有 活动对抑制也同样敏感。 花生三氟甲基酮，但iPLA2更有效 比cPLA2更能被卤代内酯自杀底物抑制。逆转录聚合酶链式反应 从纯化的胰岛b细胞和从 荧光激活法制备富A细胞群体 细胞分选显示cpla2基因在b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 在b细胞中表达的具有不同性质的PLA2酶。

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, as demonstrated by isotope dilution mass spectrometry, which may play signaling or effector roles insulin secretion. Of enzymes that catalyze phospholipid hydrolysis, islet b-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 rec ognized by anti-cPLA2 antibody. Comparison of recombinant 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 b-cells and from an a-cell-enriched population prepared by fluorescence-activated cell-sorting indicated that cPLA2 mRNA is more abundant in the b-cell population. Immunoblotting analyses indicate that islets express cPLA2-immuno-reactive 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 b-cells.