Cloning and expression of a group IV cytosolic Ca2+-dependent phospholipase A2 from rat pancreatic islets. Comparison of the expressed activity with that of an islet group VI cytosolic Ca2+-independent phospholipase A2

Zhongmin Ma, Sasanka Ramanadham, Zhiqing Hu, John Turk
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引用次数: 54

Abstract

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may play signaling or effector roles in insulin secretion. Of enzymes that catalyze phospholipid hydrolysis, islet β-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 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 β-cells and from an α-cell-enriched population prepared by fluorescence-activated cell-sorting indicated that cPLA2 mRNA is more abundant in the β-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 β-cells.

大鼠胰岛细胞内Ca2+依赖性磷脂酶A2的克隆和表达。与胰岛组胞浆Ca2+非依赖性磷脂酶A2表达活性的比较
葡萄糖刺激胰岛诱导磷脂水解和非酯化花生四烯酸的积累,这可能在胰岛素分泌中发挥信号或效应作用。在催化磷脂水解的酶中,胰岛β细胞表达低分子量分泌型磷脂酶A2 (PLA2)和第VI类不依赖于Ca2+的磷脂酶iPLA2。先前的研究表明,胰岛也表达一种被抗体识别的蛋白质,抗IV组,细胞质,Ca2+依赖性PLA2 (cPLA2)。为了进一步研究胰岛表达cPLA2的可能性,我们用识别cPLA2序列的探针筛选了一个大鼠胰岛cDNA文库,并分离了一个全长cPLA2 cDNA。大鼠胰岛cPLA2氨基酸序列与人类和小鼠cPLA2相同96%。在表达载体中转染cPLA2 cDNA的COS-7细胞可诱导Ca2+依赖性PLA2活性和抗cPLA2抗体识别的蛋白的表达。重组胰岛细胞cPLA2与转染的COS-7细胞中表达的iPLA2活性的比较表明,ATP能刺激iPLA2而非cPLA2。这两种活性对花生四烯基三氟甲基酮的抑制同样敏感,但iPLA2受到卤烯醇内酯自杀底物的抑制比cPLA2更有效。用纯化胰岛β细胞和荧光活化细胞分选制备的富集α细胞群体的RNA进行RT-PCR实验表明,cPLA2 mRNA在β细胞群体中更丰富。免疫印迹分析表明,胰岛细胞表达cpla2免疫反应蛋白,而白细胞介素-1不影响其表达。因此,cPLA2是至少三种在β细胞中表达的具有不同特性的PLA2酶之一。
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