Studies on the interaction between Escherichia coli pyruvate oxidase and a detergent activator by utilization of the fluorescence probe bis(8-p-toluidino-1-naphthalenesulfonate).
Studies on the interaction between Escherichia coli pyruvate oxidase and a detergent activator by utilization of the fluorescence probe bis(8-p-toluidino-1-naphthalenesulfonate).
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利用荧光探针双(8-对甲苯胺基-1-萘磺酸盐)研究大肠杆菌丙酮酸氧化酶与洗涤剂活化剂之间的相互作用。
DOI：
10.1021/bi00572a010
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发表时间：
1979
期刊：
影响因子：
2.9
通讯作者：
R. Gennis
中科院分区：
文献类型：
--
作者：
T. O'brien;R. Gennis
托马斯A.奥布莱恩 * 和罗伯特B。丙酮酸氧化酶是从大肠杆菌中分离的一种外周膜黄素酶。在某些脂质和洗涤剂的存在下，纯酶的体外比活性增强25倍。此外，在酶底物和辅因子丙酮酸盐和焦磷酸硫胺素（Mg 2+）存在下，蛋白质对磷脂和去污剂的亲和力显著增强。在本文中，一种新的荧光探针被用来检查蛋白质的构象变化伴随着底物还原的黄素，氧化酶的激活，和结合的洗涤剂活化剂，十二烷基硫酸盐。在十二烷基硫酸盐存在下，探针双（8-对甲苯胺基-1-萘磺酸盐）（bis-Tns）与丙酮酸氧化酶结合，在染料浓度低于1 µ时，探针荧光大幅增加。在不存在活化剂的情况下，在低染料浓度下未观察到结合。该酶结合约16个双-Tns分子，在什么似乎是疏水结合位点。底物还原黄素蛋白不仅与双-Tns结合底物还原黄素蛋白不仅与双-Tns结合
Thomas A. O’Brien* and Robert B. Gennis* abstract: Pyruvate oxidase is a peripheral membrane fla-voenzyme isolated from Escherichia coli. Thein vitro specific activity of the pure enzyme is enhanced 25-fold in the presence of certain lipids and detergents. In addition, the affinity of the protein for both phospholipids and detergents is significantly enhanced in the presence of the enzyme substrate and cofactor, pyruvate and thiamin pyrophosphate (Mg2+). In this paper a novel fluorescent probe is used to examine the protein conformational changes concomitant with substrate reduction of the flavin, activation of the oxidase, and binding of the detergent activator, dodecyl sulfate. In the presence of dodecyl sulfate, the probe bis (8-p-toluidino-l-naphthalenesulfonate)(bis-Tns) bindsto pyruvate oxidase with a resultant large increase in probe fluorescence at dye concentrations below 1 µ. No binding at low dye concentration is observed in the absence of the activator. The enzyme binds about 16 molecules of bis-Tns, in what appear to be hydrophobic binding sites. The substrate-reduced flavoprotein bindsto bis-Tns not only