BINDING OF LEUKOTRIENE-B4 AND ITS ANALOGS TO HUMAN POLYMORPHONUCLEAR LEUKOCYTE MEMBRANE-RECEPTORS
BINDING OF LEUKOTRIENE-B4 AND ITS ANALOGS TO HUMAN POLYMORPHONUCLEAR LEUKOCYTE MEMBRANE-RECEPTORS
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DOI：
10.1016/0090-6980(87)90114-6
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发表时间：
1987-06-01
影响因子：
2.9
通讯作者：
MONG, S
中科院分区：
文献类型：
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作者：
BOMALASKI, JS;MONG, S
LTB 4诱导的PMN的促炎反应包括趋化性、趋化运动、聚集和脱粒被认为是通过LTB 4与膜受体的结合而启动的。为了进一步探索这种结合的性质，我们建立了一种受体结合试验，以研究激动剂结合的结构特异性要求。采用匀浆和不连续蔗糖密度梯度纯化法富集人中性粒细胞质膜。[~ 3 H]-LTB_4与纯化膜的结合依赖于膜蛋白的浓度和孵育时间。在20度。C，结合[3 H]-LTB 4的膜受体是快速的，需要8至10分钟达到稳态，并保持稳定长达50分钟。平衡饱和结合研究表明，[3 H]-LTB 4结合高亲和力（解离常数，Kd = 1.5 nM），和低容量（密度，Bmax=40 pmol/mg蛋白）受体位点。竞争结合研究表明，LTB 4、LTB 4-差向异构体、20-OH-LTB 4、2-nor-LTB 4、6-trans-epi-LTB 4和6-trans-LTB 4与[3 H]-LTB 4受体的结合亲和力依次降低。这些类似物的平均结合亲和力（Ki）分别为2、34、58、80、1075和1275 nM。因此，与受体的最佳结合需要立体特异性5（S）、12（R）羟基、C-6处的顺式双键和全长类二十烷酸骨架。这些类似物的结合亲和力和排序效力与它们在诱导PNM趋化性中的内在激动活性相关。这些研究表明，人PMN质膜上存在高亲和力、立体选择性和特异性的LTB 4受体。
LTB4-induced proinflammatory responses in PMN including chemotaxis, chemokinesis, aggregation and degranulation are thought to be initiated through the binding of LTB4 to membrane receptors. To explore further the nature of this binding, we have established a receptor binding assay to investigate the structural specificity requirements for agonist binding. Human PMN plasma membrane was enriched by homogenization and discontinuous sucrose density gradient purification. [3H]-LTB4 binding to the purified membrane was dependent on the concentration of membrane protein and the time of incubation. At 20.degree. C, binding of [3H]-LTB4 to the membrane receptors was rapid, required 8 to 10 min to reach a steady-state and remained stable for up to 50 min. Equilibrium saturation binding studies showed that [3H]-LTB4 bound to high affinity (dissociation constant, Kd = 1.5 nM), and low capacity (density, Bmax=40 pmol/mg protein) receptor sites. Competition binding studies showed that LTB4, LTB4-epimers, 20-OH-LTB4, 2-nor-LTB4, 6-trans-epi-LTB4 and 6-trans-LTB4, in decreasing order of affinity, bound to the [3H]-LTB4 receptors. The mean binding affinities (Ki) of these analogs were 2, 34, 58, 80, 1075 and 1275 nM, respectively. Thus, optimal binding to the receptors requires stereospecific 5(S), 12(R) hydroxyl groups, a cis-double bond at C-6, and a full length eicosanoid backbone. The binding affinity and rank-order potency of these analogs correlated with their intrinsic agonistic activities in inducing PNM chemotaxis. These studies have demonstrated the existence of high affinity, stereoselective and specific receptors for LTB4 in human PMN plasma membrane.