INTERACTION OF FLUORESCENCE PROBES WITH ACETYLCHOLINESTERASE - SITE AND SPECIFICITY OF PROPIDIUM BINDING
INTERACTION OF FLUORESCENCE PROBES WITH ACETYLCHOLINESTERASE - SITE AND SPECIFICITY OF PROPIDIUM BINDING
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DOI：
10.1021/bi00680a029
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发表时间：
1975-01-01
期刊：
影响因子：
2.9
通讯作者：
LAPPI, S
中科院分区：
文献类型：
--
作者：
TAYLOR, P;LAPPI, S
已发现双季铵盐荧光探针二碘化丙啶与来自加州鱼雷的乙酰胆碱酯酶结合时，荧光强度增强十倍。该复合物的特点是具有高亲和力，μTd= 3.0 X 10-7 M，并且与乙酰胆碱酯酶的 82,000 分子量亚基呈 1:1 化学计量。多种其他季铵配体，例如十甲铵、没食子胺、rf-管箭毒碱、四乙铵和四甲铵，将丙啶从酶中完全解离，一价和二价无机阳离子也是如此。竞争性解离并不表现出合作行为或占据不同亲和力的多个位点以产生位移的明显要求。虽然可以从宏观上说明直接竞争关系，但各种四元配体对无机阳离子置换表现出不同的敏感性。丙啶相对于没食子胺的亲和力随着离子强度的增加而增加。这一发现表明，从使用天然和合成底物的稳态动力学研究来看，不完全等同，很明显，各种季铵配体和无机阳离子在多个位点与乙酰胆碱酯酶强烈相互作用（Changeux，1966；Kitz 等，1970；Wombacher 和 Wolf，1971；Belleau 等，1970）。这一证据得到了核磁共振（Kato，1972）和最近配体与乙酰胆碱酯酶关联的荧光光谱测量的加强（Mooser 等，1972；Mooser 和 Sigman，1974）。后一种方法具有固有的优势，即可以直接测量配体缔合，而不是依赖于配体缔合的影响
A bis-quaternary fluorescence probe, propidium diiodide, has been found to exhibit a tenfold enhancement of fluorescence when bound to acetylcholinesterase from Torpedo californica. The complex is characterized by a high affinity, ÁTd= 3.0 X 10-7 M, and 1: 1 stoichiometry with the 82,000 molecular weight subunit of acetylcholines-terase. A wide variety of other quaternary ammonium lig-ands such as decamethonium, gallamine, rf-tubocurarine, tetraethylammonium, and tetramethylammonium will com-pletely dissociate propidium from the enzyme as will mono-valent and divalent inorganic cations. The competitive dis-sociation does not show cooperative behavior or a distinct requirement for occupation of multiple sites of different af-finity to produce displacement. While a directly competitive relationship can be illustrated macroscopically, the various quaternary ligands show a different susceptibility toward inorganic cation displacement. The affinity of propidium relative to gallamine increases with ionic strength. This finding indicates that there is not complete equivalence inFrom steady-state kinetic studies using natural and syn-thetic substrates, it has become evident that various quaternary ammonium ligands and inorganic cations interact strongly with acetylcholinesterase at more than a single site (Changeux, 1966; Kitz et al., 1970; Wombacher and Wolf, 1971; Belleau et al., 1970). This evidence has been reinforced by nuclear magnetic resonance(Kato, 1972) and, more recently, fluorescence spectroscopicmeasurements of ligand association with acetylcholinesterase (Mooser et al., 1972; Mooser and Sigman, 1974). The latter approaches possess the inherent advantage that ligand association can be measured directly ratherthan relying upon the influence