Ligand binding properties of acetylcholinesterase determined with fluorescent probes.
Ligand binding properties of acetylcholinesterase determined with fluorescent probes.
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用荧光探针测定乙酰胆碱酯酶的配体结合特性。
DOI：
10.1021/bi00708a010
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发表时间：
1974
期刊：
影响因子：
2.9
通讯作者：
D. Sigman
中科院分区：
文献类型：
--
作者：
G. Mooser;D. Sigman
Gregory Mooser和David S.Sigman摘要：荧光探针(V-甲基吖啶离子和双(3-氨基吡啶)-L，10-癸烷)被用来监测电鳗乙酰胆碱酯酶的配体结合特性。探针被胆碱能配体置换的特征是探针荧光随置换配体浓度的增加而增加。用非线性最小二乘回归分析将数据拟合到三个配体结合模型中。结果表明，D-管胡萝卜碱和加拉胺可以结合到酶的外围配体结合部位，而不同于催化部位的底物结合部位。该酶的配体结合特性对离子强度非常敏感。当离子强度为0.16m时，β/-管胡萝卜碱仅与外周部位结合，而当离子强度为0.002 m时，该受体拮抗剂既与催化部位结合又与外周部位结合。从这些结果中得出的一个可能的结论是，至少存在两种构象不同的增溶酶。外围结合部位可能与胆碱能配体的第二个阳离子基团十氯甲烷和荧光探针双(3-氨基吡啶)-L(10-癸烷)的结合部位相同。外围位置的配基结合破坏了催化位置的结合稳定性。这两种荧光探针在检测乙酰胆碱受体蛋白的配体结合特性时都是有用的。
Gregory Mooser $ and David S. Sigman* § abstract: The fluorescent probes,(V-methylacridinium ion and bis (3-aminopyridinium)-l, 10-decane, have been used to monitor the ligand binding properties of acetylcholinesterase from electric eel. The displacement of the probes by choliner-gic ligands is characterized by an increase of probe fluorescence as a function of displacing ligand concentration. Nonlinear least-squares regression analyses have been used to fit the data to three ligandbinding models. The results indicatethat d-tubocurarine and perhaps gallamine can bind to a peripheral ligand binding site on the enzyme which is distinct from the substrate binding site at the catalytic site. The ligand binding properties of the enzyme are very sensitive to ionic strength. At an ionic strength of 0.16 m,¿/-tubocurarine binds exclusively to the peripheral site whereas at an ionic strength of 0.002 m this receptor antagonist binds both to the catalytic site and the peripheral site. One possible conclusion from these results is that at least two conformationally distinct forms of the solubilized enzyme exist. The peripheral binding site is likely the same site which binds thesecond cationic group of the cholinergic ligand, decamethonium, and the fluorescent probe, bis (3-aminopyridinium)-l, 10-decane. Ligand binding at the peripheral site destabilizes binding at the catalytic site. Both fluorescent probes should be useful in examining the ligand binding properties of the acetylcholine receptor pro-tein.