MECHANISMS THAT REGULATE THE ENZYMES THAT METABOLIZE CIS UNSATURATED FATTY ACIDS

我们对PGHS-1的研究表明，Tyr自由基是由 PGHS-1的过氧化物酶活性以及该自由基参与了 花生四烯酸代谢过程中发生的自我失活。我们的 数据表明，Tyr自由基不是活性中间体， 启动花生四烯酸氧合作用。获得对…的进一步支持 这一结论我们正在研究人类体内酪氨酸自由基的形成 PGHS-2。该酶是在原核表达和纯化后筛选出来的 天然和突变的蛋白质中所需的大量可以 完成了。一氧化氮(NO)被报道可促进前列腺素 通过直接增强PGHS活性而形成。我们已经调查了 这个问题，并发现NO是过氧化物酶的底物，但 不能提高环氧合酶活性。此外，NO也不会刺激 PGHS的表达或改变内毒素依赖的PGHS-2的表达。不可能 通过改变我们所处的cPLA2的活性或表达来起作用 目前正在学习。我们还克隆了大鼠PGHS-1和-2，并对其进行了研究 在大鼠气管细胞(EGV-6)中表达。我们观察到了一个 在完整的信使核糖核酸的基础上，异常地切下前列腺素HS-1的信使核糖核酸。 此外，我们在这些细胞中观察到TPA上调了PGHS-1的表达 在大多数细胞中，它不是由配体上调的，而是结构性的 受监管的。这可能与EGV-6的变形性质有关 细胞。我们已经证明了EGF调节13-HODE的形成。 叙利亚仓鼠胚胎(SHE)细胞。投入了相当大的努力来 克隆这种表面上的15-脂氧合酶，试图开发工具 有必要研究EGF对其的调控(见细胞增殖 项目)。尽管付出了相当大的努力并采取了几种方法，我们还是 无法克隆这种15-脂氧合酶。这表明这种酶是 与任何特征的脂氧合酶无关。我们成功地做到了 克隆、测序并在大肠杆菌中表达仓鼠5-脂氧合酶。我们的 了解EGF对13-HODE形成的调控将 转向也能代谢亚油酸的人乳腺细胞培养 至13-HODE，并受EGF监管。必要的工具可用于 人类的酵素。

Our studies with PGHS-1 indicate that a Tyr radical is formed by the peroxidase activity of PGHS-1 and that this radical is involved in the self-inactivation that occurs during arachidonic acid metabolism. Our data suggest that the Tyr radical is not the reactive intermediate that initiates arachidonic acid oxygenation. To obtain further support for this conclusion we are examining the formation of Tyr radical in human PGHS-2. This enzyme was selected since expression and purification of the native and mutants proteins in the necessary large amounts can be accomplished. Nitric oxide(NO) is reported to enhance prostaglandin formation by direct enhancement of PGHS activity. We have investigated this problem and found that NO is a substrate for the peroxidase but does not enhance the cyclooxygenase activity. Also, NO does not stimulate the expression of PGHS or alter LPS dependent PGHS-2 expression. NO could act by altering the activity or expression of cPLA2 which we are currently studying. We have also cloned rat PGHS-1 and -2 and studied their expression by rat tracheal cells (EGV-6). We observed an aberrantly sliced PGHS-1 mRNA in addition to the complete mRNA. Furthermore, we observed in these cells that TPA up-regulated PGHS-1 which in most cells is not upregulated by ligands but constitutively regulated. This may be related to the transformed nature of the EGV-6 cells. We have shown that EGF regulates the formation of 13-HODE in Syrian hamster embryo (SHE) cells. Considerable effort was devoted to cloning this apparent 15-lipoxygenase in an attempt to develop the tools necessary to investigate its regulation by EGF (see cell proliferation project). Despite considerable effort and several approaches, we were unable to clone this 15-lipoxygenase. This suggests that the enzyme is not related to any characterized lipoxygenase. We did successfully clone, sequence and express in, E. coli, a hamster 5-lipoxygenase. Our efforts to understand the regulation by EGF of 13-HODE formation will shift to human breast cell cultures which also metabolize linoleic acid to 13-HODE and is regulated by EGF. The necessary tools are available for the human enzyme.