Methods for studying synaptosomal copper release
Methods for studying synaptosomal copper release
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DOI：
10.1016/s0165-0270(03)00173-0
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发表时间：
2003-09-30
影响因子：
3
通讯作者：
Herms, J
中科院分区：
文献类型：
--
作者：
Hopt, A;Korte, S;Herms, J
Cu被认为在多种神经退行性疾病的发病机制中发挥着重要作用，例如威尔逊氏病、阿尔茨海默氏病，并且可能在克雅氏病等朊病毒蛋白疾病中发挥重要作用。到目前为止，还没有方法可以确定体内这种阳离子的浓度。在这里，我们提出了两种可能的方法，并对结果进行了严格的比较。近年来，荧光配体成功用于测定 Ca2+ 浓度，这鼓励我们寻找一种与 Cu2+ 特异性反应的荧光团，并根据我们的目的对其进行表征。我们发现 TSPP（四（4-磺基苯基）卟啉）在 645 nm 发射波长下的体外发射对 Cu2+ 具有高度特异性（表观解离常数 K-d = 0.43 +/- 0.07 muM，pH 7.4）。与大多数其他检查的染料不同，它不会与大脑中最常见的二价阳离子 Ca2+ 和 Mg2+ 发生反应。此外，Zn2+ 在不同的发射波长 (605 nm) 下猝灭 TSPP 荧光，K-d 为 50 +/- 2.5 muM (pH 7.0)。有了这些发现，我们将 TSPP 的铜测量应用于生物系统，首次在体内表明去极化时突触体会释放铜。我们的研究结果通过基于 ICP-MS（电感耦合等离子体质谱）的完全独立的分析方法进行了验证。 (C) 2003 Elsevier B.V. 保留所有权利。
Cu is thought to play an important role in the pathogenesis of several neurodegenerative diseases, such as Wilson's, Alzheimer's, and probably in prion protein diseases like Creutzfeld-Jakob's disease. Until now, no method existed to determine the concentration of this cation in vivo. Here, we present two possible approaches combined with a critical comparison of the results. The successful use of fluorescent ligands for the determination of Ca2+-concentrations in recent years encouraged us to seek a fluorophore which specifically reacts to Cu2+ and to characterize it for our purposes. We found that the emission of TSPP (tetrakis(4-sulfophenyl)porphine) at an emission wavelength of 645 nm is in vitro highly specific to Cu2+ (apparent dissociation constant K-d = 0.43 +/- 0.07 muM at pH 7.4). It does not react with the most common divalent cations in the brain, Ca2+ and Mg2+, unlike most of the other dyes examined. In addition, Zn2+ quenches TSPP fluorescence at a different emission wavelength (605 nm) with a K-d of 50 +/- 2.5 muM (pH 7.0). With these findings, we applied the measurement of Cu with TSPP to a biological system, showing for the first time in vivo that there is release of copper by synaptosomes upon depolarisation. Our findings were validated with a completely independent analytical approach based on ICP-MS (inductively-coupled-plasma mass-spectrometry). (C) 2003 Elsevier B.V. All rights reserved.