Studies on Leukotriene Biosynthesis and Signal Transduction.

PAF和白三烯是参与炎症/过敏反应以及各种心血管和免疫紊乱的脂质介质。我们对这些脂质介体的生物合成和信号转导进行了实验。PAF和白三烯受体的分离：通过非洲爪哇卵母细胞系统的表达克隆，我们获得了PAF受体的一个cDNAs，这是第一个成功克隆脂质介质受体的例子。还获得了人类同源物。它们由342个氨基酸残基组成，推测有7个跨膜结构域。这些受体在COS细胞或CHO细胞中表达，细胞信号转导得到了广泛的研究。结果表明，PAF受体通过不同类型的G蛋白与磷脂酶、腺苷环化酶和MAP激酶偶联。白三烯受体(B4和D4)分别从豚鼠白细胞和肺膜上以活性形式溶解。它们通过Gi蛋白激活磷脂酶A_2和C。…通过两种策略(蛋白质纯化和表达克隆)对这些受体进行更多的分子克隆目前正在进行中。白三烯A_4水解酶。白三烯B_4是一种双功能酶。白三烯B_4是一种有效的趋化因子，由一种名为白三烯A4水解酶的酶产生。通过对该酶的cDNA克隆和一级结构的阐明，表明该酶与多种类型的金属蛋白酶具有高度的同源性。事实上，该酶含有锌离子，这是内源性氨基肽酶活性和白三烯A_4水解酶所必需的。为了确定酶的活性部位，进行了定点诱变实验。His-296或Glu-315突变导致锌离子丢失，并伴随着两种酶活性的消失。然而，通过将Glu-297改变为其他氨基酸。两种酶的活性明显分离。水分子的掺入机制以及酶的双功能作用的生理意义尚不清楚。较少

PAF and leukotrienes are lipid mediators involved in inflammatory/allergic response as well as various cardiovascular and immunological disorders. We have carried out experiments on biosyntheses and signal transduction of these lipid mediators.1. Isolation of PAF and leukotriene receptors.By expression cloning using Xenopus oocyte system, we isolated a cDNA of PAF receptor as the first successful example of receptor cloning of lipid mediators. A human homologue was also obtained. They are composed of 342 amino acid residues with putative seven transmembrane spanning domains. These receptors were expressed in COS cells or CHO cells, and cellular signalings are extensively studied. It was shown that PAF receptor couples to phospholipases, adenylate cyclase, and MAP kinase via different types of G- proteins. Leukotriene receptors (B4, and D4) were solubilized from guinea pig leukocyte and lung membrane, respectively in an active form. They activate phospholipase A_2 and C via Gi proteins. … More Molecular cloning of these receptors by two strategies (protein purification and expression cloning) is currently ongoing.2. Leukotriene A_4 hydrolase. a bifunctional enzyme.Leukotriene B_4, a potent chemotactic factor, is produced by an enzyme termed leukotriene A4 hydrolase. By cDNA cloning and elucidation of the primary structure of the enzyme have shown that the enzyme is highly homologous to various types of metalloproteinases. In fact, it was shown that the enzyme contains a zinc ion, which is essential for both intrinsic aminopeptidase activity and leukotriene A_4 hydrolase. To determine the active site of the enzyme, site-directed mutagenesis experiments were performed. The mutation of His-296, or Glu-315 yielded loss of zinc ion with concomitant disappearance of both enzyme activities. However, by changing Glu-297 to other amino acids. two enzyme activities were clearly separated. The mechanism of incorporation of water molecule as well as the physiological significance of bifunctional role of enzyme remain unclear. Less