Brock T G, Paine R, Peters-Golden M
Department of Internal Medicine, University of Michigan, Ann Arbor.
J Biol Chem. 1994 Sep 2;269(35):22059-66.
Arachidonate metabolism by 5-lipoxygenase (5-LO) coincides with the translocation of the enzyme from a soluble to a pelletable fraction in thoroughly disrupted granulocytic cells. While immunoelectron microscopy has identified the nuclear membrane as the site at which 5-LO, as well as 5-LO activating protein (FLAP), are localized in activated cells, the locale of soluble 5-LO in unstimulated cells could not be established by this technique. We asked whether the nucleus might also be the site for soluble 5-LO in unstimulated cells, and utilized rat basophilic leukemia (RBL) cells as model granulocytic cells to address this question. Using three different techniques to disrupt cells while leaving nuclei intact (mild nitrogen cavitation, Dounce homogenization, and detergent lysis), immunoblot analysis indicated abundant 5-LO in isolated nuclei. Within purified nuclei, 5-LO existed in two pools: a soluble pool that was readily released upon nuclear disruption and a bound pool that was not removed by 300 mM NaCl treatment. In all cases, 5-LO was also found in cytosolic and non-nuclear membrane fractions. Indirect immunofluorescent microscopy confirmed the presence of abundant 5-LO within the nucleus with minimal extranuclear signal in most cells. However, a minority of cells, characterized by condensed chromatin, showed no nuclear-associated staining with increased cytoplasmic staining for 5-LO. This suggested that some of the cytosolic 5-LO found by cell fractionation resulted from these dividing cells. When the contribution from dividing cells was minimized, either by overnight serum deprivation or by isolating cytoplasts of nucleus-containing cells, 5-LO was prominent in the nuclear fraction but negligible in the cytosolic fraction. In contrast to this distribution in RBL cells, 5-LO in unstimulated human neutrophils was predominantly cytosolic, by both immunoblot and immunofluorescence analyses. In both RBL cells and human neutrophils, FLAP was localized at the nuclear membrane and the endoplasmic reticulum. These data provide the first evidence for the localization of 5-LO in unstimulated granulocytic cells. The finding that a substantial proportion of enzyme is localized within the nucleus of unstimulated RBL cells suggests potentially novel roles for 5-LO or its products within the nucleus.
在完全破碎的粒细胞中，5-脂氧合酶（5-LO）催化的花生四烯酸代谢与该酶从可溶性组分向可沉淀组分的转位相吻合。虽然免疫电子显微镜已确定核膜是活化细胞中5-LO以及5-LO激活蛋白（FLAP）的定位位点，但该技术无法确定未刺激细胞中可溶性5-LO的定位。我们研究了细胞核是否也是未刺激细胞中可溶性5-LO的定位位点，并利用大鼠嗜碱性白血病（RBL）细胞作为粒细胞模型来解决这个问题。使用三种不同的技术在保持细胞核完整的情况下破碎细胞（温和氮气空化、Dounce匀浆和去污剂裂解），免疫印迹分析表明分离的细胞核中有大量5-LO。在纯化的细胞核中，5-LO存在于两个池中：一个是在核破碎时容易释放的可溶性池，另一个是经300 mM NaCl处理后未被去除的结合池。在所有情况下，在细胞质和非核膜组分中也发现了5-LO。间接免疫荧光显微镜证实大多数细胞的细胞核内存在大量5-LO，核外信号极少。然而，少数以染色质凝聚为特征的细胞，未显示与细胞核相关的染色，而5-LO的细胞质染色增加。这表明细胞分级分离发现的一些细胞质5-LO来自这些分裂细胞。当通过过夜血清剥夺或分离含细胞核细胞的胞质体将分裂细胞的贡献最小化时，5-LO在细胞核组分中很突出，但在细胞质组分中可忽略不计。与RBL细胞中的这种分布不同，通过免疫印迹和免疫荧光分析，未刺激的人类中性粒细胞中的5-LO主要位于细胞质中。在RBL细胞和人类中性粒细胞中，FLAP均定位于核膜和内质网。这些数据首次证明了5-LO在未刺激的粒细胞中的定位。未刺激的RBL细胞中相当一部分酶定位于细胞核这一发现表明5-LO或其产物在细胞核内可能具有新的作用。
