Coffey M, Peters-Golden M, Fantone J C, Sporn P H
Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109.
J Biol Chem. 1992 Jan 5;267(1):570-6.
The enzyme 5-lipoxygenase (5-LO) catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes, substances which play pivotal roles both in normal host defense and in pathologic states of inflammation. Recent studies in granulocytic cells have shown that activation of 5-LO involves its Ca(2+)-dependent translocation from cytosol to membrane compartments. However, little information exists about the molecular regulation of 5-LO in macrophages, even though these cells comprise the resident effector cell population of most organs. We therefore examined the levels of 5-LO activity and immunoreactive protein in cytosol and membrane fractions of resident rat alveolar (AM) and peritoneal macrophages (PM) and compared them with the well studied human neutrophil (polymorphonuclear leukocyte). In the resting state, PM resembled polymorphonuclear leukocyte in that most of their cell-free 5-LO activity, as well as protein content, were localized to the cytosol fraction. By contrast, resting AM contained most of their activity and almost half of their immunoreactive protein in the crude membrane fraction. The inability of the drug MK-886 to reverse this membrane association suggested that the 5-LO-activating protein was not the site of binding in the resting cell; however, this drug completely inhibited leukotriene B4 synthesis in ionophore A23187-stimulated AM, indicating that an interaction between 5-LO and 5-LO-activating protein was nonetheless required for product synthesis upon stimulation. Translocation of cytosolic 5-LO protein could not be convincingly demonstrated in A23187-stimulated AM, suggesting that the pool of 5-LO enzyme responsible for product formation originated in the membrane rather than the cytosol fraction of the resting cell. The AM therefore represents the first mammalian cell in which 5-LO has been recovered from the membrane fraction (a) of a resting cell and (b) in active form. These novel findings extend our understanding of the molecular regulation of 5-LO and may be of importance in designing strategies to limit inflammation in the lung and other sites.
5-脂氧合酶（5-LO）催化花生四烯酸代谢生成白三烯的前两个步骤，白三烯在正常宿主防御和炎症病理状态中均起关键作用。近期对粒细胞的研究表明，5-LO的激活涉及其依赖钙离子从胞质溶胶向膜区室的转位。然而，尽管巨噬细胞是大多数器官中的常驻效应细胞群体，但关于巨噬细胞中5-LO的分子调控信息却很少。因此，我们检测了大鼠常驻肺泡巨噬细胞（AM）和腹腔巨噬细胞（PM）的胞质溶胶和膜组分中5-LO活性水平及免疫反应性蛋白，并将其与研究充分的人类中性粒细胞（多形核白细胞）进行比较。在静息状态下，PM与多形核白细胞相似，其大部分无细胞5-LO活性以及蛋白质含量都定位于胞质溶胶组分。相比之下，静息AM的大部分活性以及近一半的免疫反应性蛋白存在于粗膜组分中。药物MK-886无法逆转这种膜结合，这表明5-LO激活蛋白不是静息细胞中的结合位点；然而，该药物完全抑制了离子载体A23187刺激的AM中白三烯B4的合成，这表明在刺激时产物合成仍需要5-LO与5-LO激活蛋白之间的相互作用。在A23187刺激的AM中无法令人信服地证明胞质5-LO蛋白的转位，这表明负责产物形成的5-LO酶库起源于静息细胞的膜而非胞质溶胶组分。因此，AM代表了首个从静息细胞的膜组分中（a）以活性形式回收5-LO的哺乳动物细胞。这些新发现扩展了我们对5-LO分子调控的理解，可能对设计限制肺部和其他部位炎症的策略具有重要意义。
