Interaction of enkephalin with opiate receptors in intact cultured cells.
Interaction of enkephalin with opiate receptors in intact cultured cells.
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完整培养细胞中脑啡肽与阿片受体的相互作用。
DOI：
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发表时间：
1978
影响因子：
3.6
通讯作者：
P. Cuatrecasas
中科院分区：
文献类型：
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作者：
K. Chang;R. Miller;P. Cuatrecasas
用代谢稳定的脑啡肽类似物(D-Ala2，D-Leu5)-脑啡肽的高比活性衍生物(125I标记)，研究了脑啡肽与完整神经母细胞瘤细胞(N4TG1)中阿片受体的相互作用。低浓度的天然(Leu5)-和(Met5)-脑啡肽和β-内啡肽可以特异性地抑制这种结合，但β-促脂激素不能抑制这种结合。结合数据清楚地显示了单一类别的均一结合位点，没有证据表明在任何配体浓度下存在协同作用。在24°时，与细胞结合的结合和解离速率常数分别为3×107M-1min-1和0.035 min-1。在细胞中，当每106个细胞结合30个分子脑啡肽时，[125I](D-Ala2，D-Leu5)-脑啡肽的结合达到饱和。Kd为1~2 nm。37°孵育未见受体或配体-受体复合体内化的证据。完整细胞中的受体非常抵抗蛋白酶和磷脂酶A的消化，这与膜制剂中观察到的结果形成鲜明对比，膜制剂对这些酶表现出极高的敏感性。这种抗性显然不是由于受体池在细胞内的定位，因为细胞匀浆与完整细胞具有相同数量的受体位置。在细胞中，脑啡肽受体的亲和力对介质的阳离子成分非常敏感。在Na+存在的情况下，去除二价阳离子Mg2+和Ca2+，亲和力降低约3.5倍。麻醉性激动剂和拮抗剂比[125I](D-Ala2，D-Leu5)-脑啡肽结合更能抑制[~3H]纳洛酮结合。脑啡肽的情况正好相反，尽管依托吗啡和β-内啡肽与两种标记的配体竞争同样好。这些结果表明，阿片剂和脑啡肽与同一受体的结合方式不同，或与不同受体的结合具有重叠的特异性。
The interaction of enkephalins with opiate receptors in intact neuroblastoma cells (N4TG1) was studied by the use of a derivative of high specific activity (125I-labeled) of the metabolically stable enkephalin analogue, (D-Ala2, D-Leu5)-enkephalin. The binding is specifically inhibited by low concentrations of the natural (Leu5)- and (Met5)-enkephalins and by β-endorphin, but not by β-lipotropin. The binding data clearly show a single class of homogeneous binding sites without evidence for cooperative interactions at any ligand concentration. The association and dissociation rate constants of binding to cells are 3 x 107 M-1min-1 and 0.035 min-1, respectively, at 24°. In cells, the binding of [125I] (D-Ala2, D-Leu5)-enkephalin is saturated when 30 fmoles of the peptide are bound per 106 cells. The Kd is 1 to 2 nM. No evidence for receptor or ligand-receptor complex internalization could be demonstrated on incubating the cells at 37°. The receptors in intact cells are very resistant to digestion by proteases and phospholipase A, in sharp contrast to the results observed in membrane preparations which show exquisite sensitivity to these enzymes. This resistance is apparently not due to the intracellular localization of a pool of receptors since cell homogenates have the same number of receptor sites as intact cells. In cells, the affinity of the receptor for enkephalin is very sensitive to the cationic composition of the medium. Removal of the divalent cations, Mg2+ and Ca2+, in the presence of Na+ reduces the affinity by about 3.5-fold. Narcotic agonists and antagonists are more potent in inhibiting [3H]naloxone binding than [125I] (D-Ala2, D-Leu5)-enkephalin binding. The opposite is true for enkephalins, although etorphine and β-endorphin compete with both labeled ligands equally well. These results suggest that opiates and enkephalins bind differently to the same receptor or bind to different receptors with overlapping specificity.