Falcone R C, Aharony D
Department of Pharmacology, ICI Americas, Inc., Wilmington, Delaware.
J Pharmacol Exp Ther. 1990 Nov;255(2):565-71.
We investigated the mechanism and modulation of 3H-labeled leukotriene B4 (LTB4) binding to guinea pig lung membranes. [3H] LTB4 bound specifically and rapidly (Kobs = 0.06 +/- 0.006 min-1) to high affinity (Kd = 0.63 +/- 0.06 nM) and saturable (Bmax = 224 +/- 16 fmol/mg) sites without cooperativity (nH = 1.05). Bound ligand was partially (70%) dissociated with excess LTB4 or the selective antagonist U-75,302. Complete dissociation could be achieved with either LTB4 or U-75,302 in combination with 10 microM GTP gamma S. A series of structural analogs and selective antagonists inhibited binding in a competitive manner with the following order: LTB4 much greater than 20-(OH)-LTB4 greater than LTB5 greater than LTB-dimethylamide = U-75,302 greater than 12(R)-hydroxy-eicosatetraenoic acid greater than 5(S),12(S)-dihydroxy,6-trans,8-cis,10-trans,14-cis-ETE greater than 12(S)-hydroxy-eicosatetraenoic acid much greater than trans-LTB4 isomers. 5(S)-hydroxy-eicosatetraenoic acid, prostaglandins, peptide-leukotrienes and platelet-activating factor (at 10 microM each) did not inhibit, providing evidence that [3H]LTB4 bound to specific receptors on guinea pig lung membranes. N-Ethylmaleimide (NEM) and p-chloromercuriphenyl sulfonic acid (PCMP) inhibited binding (IC50 = 144 +/- 66 and 4.6 +/- 1.0 microM, respectively) in an irreversible manner, as evident by an inability to overcome the inhibition by extensive washing.(ABSTRACT TRUNCATED AT 250 WORDS)
我们研究了3H标记的白三烯B4（LTB4）与豚鼠肺膜结合的机制及调节情况。[3H]LTB4特异性且快速地（观测速率常数Kobs = 0.06 ± 0.006分钟-1）与高亲和力（解离常数Kd = 0.63 ± 0.06 nM）和可饱和（最大结合量Bmax = 224 ± 16 fmol/mg）的位点结合，不存在协同性（Hill系数nH = 1.05）。结合的配体可被过量的LTB4或选择性拮抗剂U - 75,302部分（70%）解离。LTB4或U - 75,302与10 microM的鸟苷三磷酸γ - 硫酯（GTPγS）联合使用可实现完全解离。一系列结构类似物和选择性拮抗剂以竞争性方式抑制结合，顺序如下：LTB4远大于20 -（OH）- LTB4大于LTB5大于LTB - 二甲酰胺 = U - 75,302大于12（R）- 羟基 - 二十碳四烯酸大于5（S），12（S）- 二羟基，6 - 反式，8 - 顺式，10 - 反式，14 - 顺式 - 二十碳四烯酸大于12（S）- 羟基 - 二十碳四烯酸远大于反式 - LTB4异构体。5（S）- 羟基 - 二十碳四烯酸、前列腺素、肽类白三烯和血小板活化因子（各为10 microM）均无抑制作用，这表明[3H]LTB4与豚鼠肺膜上的特异性受体结合。N - 乙基马来酰亚胺（NEM）和对氯汞苯磺酸（PCMP）以不可逆方式抑制结合（半数抑制浓度IC50分别为144 ± 66和4.6 ± 1.0 microM），大量洗涤无法克服这种抑制作用即可证明。（摘要截短于250字）
