CLONING & EXPRESSION OF GROUP IV CYTOSOLIC CA2+ DEPENDENT PHOSPHOLIPASE

用葡萄糖刺激胰岛诱导磷脂 非酯化花生四烯酸水解和积累， 可能在胰岛素分泌中起信号或效应作用。 酶 催化磷脂水解的酶，胰岛？细胞表达低 分子量分泌型磷脂酶A2（PLA 2）和VI组， Ca 2+非依赖性PLA 2（iPLA 2）。 先前的研究表明， 还表达被抗IV组抗体识别的蛋白质， 胞浆型钙离子依赖型磷脂酶A2（cPLA 2）。 进一步审查 为了研究cPLA 2在胰岛中的可能表达，我们筛选了大鼠胰岛cDNA 用识别cPLA 2序列的探针构建了一个文库， 全长cPLA 2 cDNA。 大鼠胰岛cPLA 2推导的氨基酸 序列与人和小鼠cPLA 2的序列96%相同。 用表达载体转染cPLA 2 cDNA的COS-7细胞 Ca 2+依赖性PLA 2活性和蛋白质的诱导表达 被抗cPLA 2抗体识别。 比较 表达的重组胰岛cPLA 2和iPLA 2活性， 转染的COS-7细胞表明，iPLA 2而不是cPLA 2是 由ATP刺激。 这两种活动都对 花生四烯基三氟甲基酮的抑制作用，但iPLA 2更强 有效抑制卤烯醇内酯硅化物底物， cPLA2。 用纯化的胰岛RNA进行RT-PCR实验？-细胞和 从一个？通过荧光激活的细胞富集的群体制备 细胞分选表明，cPLA 2 mRNA在？细胞 人口 免疫印迹分析表明，胰岛表达 cPLA 2免疫反应蛋白，白细胞介素-1不影响 它的表达。 因此，cPLA 2是至少三类细胞中的一种。 具有不同特性的PLA 2酶在？-细胞

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.