Woods J W, Coffey M J, Brock T G, Singer I I, Peters-Golden M
Department of Biochemical and Molecular Pathology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
J Clin Invest. 1995 May;95(5):2035-46. doi: 10.1172/JCI117889.
5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are two key proteins involved in the synthesis of leukotrienes (LT) from arachidonic acid. Although both alveolar macrophages (AM) and peripheral blood leukocytes (PBL) produce large amounts of LT after activation, 5-LO translocates from a soluble pool to a particulate fraction upon activation of PBL, but is contained in the particulate fraction in AM irrespective of activation. We have therefore examined the subcellular localization of 5-LO in autologous human AM and PBL collected from normal donors. While immunogold electron microscopy demonstrated little 5-LO in resting PBL, resting AM exhibited abundant 5-LO epitopes in the euchromatin region of the nucleus. The presence of substantial quantities of 5-LO in the nucleus of resting AM was verified by cell fractionation and immunoblot analysis and by indirect immunofluorescence microscopy. In both AM and PBL activated by A23187, all of the observable 5-LO immunogold labeling was found associated with the nuclear envelope. In resting cells of both types, FLAP was predominantly associated with the nuclear envelope, and its localization was not affected by activation with A23187. The effects of MK-886, which binds to FLAP, were examined in ionophore-stimulated AM and PBL. Although MK-886 inhibited LT synthesis in both cell types, it failed to prevent the translocation of 5-LO to the nuclear envelope. These results indicate that the nuclear envelope is the site at which 5-LO interacts with FLAP and arachidonic acid to catalyze LT synthesis in activated AM as well as PBL, and that in resting AM the euchromatin region of the nucleus is the predominant source of the translocated enzyme. In addition, LT synthesis is a two-step process consisting of FLAP-independent translocation of 5-LO to the nuclear envelope followed by the FLAP-dependent activation of the enzyme.
5-脂氧合酶（5-LO）和5-脂氧合酶激活蛋白（FLAP）是参与从花生四烯酸合成白三烯（LT）的两种关键蛋白。尽管肺泡巨噬细胞（AM）和外周血白细胞（PBL）在激活后都会产生大量的LT，但5-LO在PBL激活后会从可溶性池转移到颗粒部分，而在AM中，无论是否激活，5-LO都存在于颗粒部分。因此，我们研究了从正常供体采集的自体人AM和PBL中5-LO的亚细胞定位。免疫金电子显微镜显示静息PBL中几乎没有5-LO，而静息AM在细胞核的常染色质区域表现出丰富的5-LO表位。通过细胞分级分离和免疫印迹分析以及间接免疫荧光显微镜证实了静息AM细胞核中存在大量的5-LO。在由A23187激活的AM和PBL中，所有可观察到的5-LO免疫金标记都与核膜相关。在两种类型的静息细胞中，FLAP主要与核膜相关，其定位不受A23187激活的影响。在离子载体刺激的AM和PBL中研究了与FLAP结合的MK-886的作用。尽管MK-886在两种细胞类型中均抑制LT合成，但它未能阻止5-LO向核膜的转移。这些结果表明，核膜是5-LO与FLAP和花生四烯酸相互作用以催化激活的AM以及PBL中LT合成的位点，并且在静息AM中，细胞核的常染色质区域是转移酶的主要来源。此外，LT合成是一个两步过程，包括5-LO不依赖FLAP向核膜的转移，随后是FLAP依赖的酶激活。
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