Chau L Y, Hoover R L, Austen K F, Lewis R A
J Immunol. 1986 Sep 15;137(6):1985-92.
The subcellular distribution of specific binding sites for [3H]leukotriene C4 ([3H]LTC4) was analyzed after sedimentation of organelles from disrupted bovine aortic endothelial cells on sucrose density gradients and was shown to be in membrane fractions I (20% sucrose) and IV (35% sucrose). Saturation binding studies of [3H]LTC4 on endothelial cell monolayers at 4 degrees C demonstrated high-affinity binding sites with a dissociation constant (Kd) of 6.8 +/- 2.2 nM (mean +/- SD) and a density of 0.12 +/- 0.02 pmol/10(6) cells. At 4 degrees C, the specific binding of [3H]LTC4 by each of the subcellular fractions reached equilibrium at 30 min and remained stable for an additional 60 min. After 30 min of incubation with [3H]LTC4, the addition of excess unlabeled LTC4 to each subcellular fraction reversed more than 70% of [3H]LTC4 binding in 10 min. The [3H]LTC4 binding activities of subcellular fractions were enhanced approximately twofold to fourfold in the presence of Ca2+, Mg2+, and Mn2+, whereas Na+, K+, and Li+ were without effect. As measured by saturation experiments, the Kd and density of LTC4 binding sites in fraction I were 4.8 +/- 1.6 nM and 16.5 +/- 1.9 pmol/mg of protein, respectively, and in fraction IV were 4.7 +/- 1.5 nM and 81.4 +/- 19 pmol/mg of protein, respectively. Inhibition of [3H]LTC4 binding in membrane-enriched subcellular fractions I and IV by LTC4 occurred with molar inhibition constant (Ki) values of 4.5 +/- 0.1 nM and 4.7 +/- 1.2 nM, respectively, whereas Ki values for LTD4 were 570 +/- 330 nM and 62.5 +/- 32.8 nM, respectively, and for LTE4 were greater than 1000 nM for each fraction; LTB4 and reduced glutathione were even less active. FPL55712, a putative antagonist of the sulfidopeptide LT components of slow reacting substance of anaphylaxis, had Ki values of 1520 +/- 800 nM and 1180 +/- 720 nM for [3H]LTC4 binding sites on membrane-enriched subcellular fractions I and IV, respectively. Thus as defined by Kd, Ki, and specificity, the LTC4 binding units that are distributed to the plasma membrane and the binding units in the subcellular fraction of greater density were similar to each other. Pretreatment of the isolated subcellular membrane fractions with trypsin abolished [3H]LTC4 binding by fraction I, enriched for the plasma membrane marker 5' nucleotidase, and that by fraction IV, enriched for the mitochondrial membrane marker succinate-cytochrome C reductase.(ABSTRACT TRUNCATED AT 400 WORDS)
在蔗糖密度梯度上对破碎的牛主动脉内皮细胞的细胞器进行沉降后，分析了[3H]白三烯C4（[3H]LTC4）特异性结合位点的亚细胞分布，结果显示其存在于膜组分I（20%蔗糖）和IV（35%蔗糖）中。在4℃下对内皮细胞单层进行[3H]LTC4的饱和结合研究，结果表明存在高亲和力结合位点，解离常数（Kd）为6.8±2.2 nM（平均值±标准差），密度为0.12±0.02 pmol/10(6)个细胞。在4℃时，每个亚细胞组分对[3H]LTC4的特异性结合在30分钟时达到平衡，并在另外60分钟内保持稳定。用[3H]LTC4孵育30分钟后，向每个亚细胞组分中加入过量未标记的LTC4，可在10分钟内使[3H]LTC4结合的70%以上被逆转。在存在Ca2+、Mg2+和Mn2+的情况下，亚细胞组分的[3H]LTC4结合活性增强约2至4倍，而Na+、K+和Li+则无作用。通过饱和实验测量，组分I中LTC4结合位点的Kd和密度分别为4.8±1.6 nM和16.5±1.9 pmol/mg蛋白质，组分IV中分别为4.7±1.5 nM和81.4±19 pmol/mg蛋白质。LTC4对富含膜的亚细胞组分I和IV中[3H]LTC4结合的抑制作用，其摩尔抑制常数（Ki）值分别为4.5±0.1 nM和4.7±1.2 nM，而LTD4的Ki值分别为570±330 nM和62.5±32.8 nM，每个组分中LTE4的Ki值均大于1000 nM；LTB4和还原型谷胱甘肽的活性更低。FPL55712是一种推测的过敏反应慢反应物质的硫肽白三烯组分的拮抗剂，其对富含膜的亚细胞组分I和IV上[3H]LTC4结合位点的Ki值分别为1520±800 nM和1180±720 nM。因此，根据Kd、Ki和特异性定义，分布于质膜的LTC4结合单位与密度较大的亚细胞组分中的结合单位彼此相似。用胰蛋白酶预处理分离的亚细胞膜组分，可消除富含质膜标志物5'核苷酸酶的组分I和富含线粒体膜标志物琥珀酸-细胞色素C还原酶的组分IV对[3H]LTC4的结合。（摘要截于400字）
