Jimenez Del Rio M, Velez Pardo C, Pinxteren J, De Potter W, Ebinger G, Vauquelin G
Department of Protein Chemistry, Free University Brussels, Genesius-Rode, Belgium.
Eur J Pharmacol. 1993 Sep 15;247(1):11-21. doi: 10.1016/0922-4106(93)90132-s.
Binding of [3H]serotonin and of [3H]dopamine to serotonin binding proteins (SBP) from soluble extracts of bovine frontal cortex is increased by Fe2+ but not by Fe3+. It was generally believed that Fe2+ first binds to sulfhydryl groups of SBP and that the monoamines form coordination bonds with the trapped iron. We report two series of findings that are incompatible with this mechanism. First, the binding of both radioligands is an irreversible process since it is not diminished when a large excess (1 mM) of serotonin or dopamine is added to a pre-equilibrated mixture of SBP, 0.1 mM Fe2+ and 0.2 microM radioligand. Once formed, binding is not impaired by chelating agents such as ethyleneglycoltetraacetic acid and desferal. Second, the Fe(2+)-stimulated binding is inhibited by reducing agents (sodium ascorbate, vitamin E, sodium metabisulfite) and by agents which deplete superoxide radicals (superoxide dismutase and hydrogen peroxide). Moreover, the effect of Fe2+ can be mimicked by oxidants (sodium periodate, potassium superoxide) and by the generation of superoxide radicals by the xanthine oxidase-catalysed oxidation of xanthine. To integrate these findings, we formulate the hypothesis that Fe2+ reacts with dissolved molecular oxygen to produce superoxide radicals, that these radicals oxidise [3H]serotonin and [3H]dopamine, and that the formed oxidation products bind covalently to cysteine residues of SBP. This alternative mechanism is also based on the ability of reagents which contain or modify sulfhydryl groups to decrease the binding and on the inability of hydroxyl radical scavengers (dimethyl sulfoxide, mannitol, ethanol and thiourea) to do so. Fe2+ is also able to irreversibly inactivate part of the binding sites on SBP (81% of the specific binding of [3H]serotonin, and 61% for [3H]dopamine). This Fe(2+)-mediated inactivation, as well as the covalent nature of the binding, preclude the interpretation of saturation and competition binding data in terms of reversible bimolecular interactions. Yet, such experiments indicate that, at the same concentration, [3H]dopamine binds to 2 to 3 times more sites than [3H]serotonin. Unlabelled dopamine acts also as a potent competitor at all the [3H]serotonin binding sites, whereas unlabelled serotonin only acts as a potent competitor at part (30%) of the [3H]dopamine binding sites. SBP were initially proposed to be involved in the storage, protection and/or transport of serotonin, and recently also of catecholamines. However, these potential functions of SBP can hardly be reconciled with the molecular mechanism of the binding. Moreover, it is conceivable that this binding actually represents an in vitro model for neurodegeneration.
来自牛额叶皮质可溶性提取物的5-羟色胺结合蛋白（SBP）对[3H]5-羟色胺和[3H]多巴胺的结合可被Fe2+增强，但不能被Fe3+增强。人们普遍认为，Fe2+首先与SBP的巯基结合，然后单胺类物质与被捕获的铁形成配位键。我们报告了两个与该机制不相符的系列研究结果。首先，两种放射性配体的结合是一个不可逆过程，因为当向预先平衡的SBP、0.1 mM Fe2+和0.2 μM放射性配体的混合物中加入大量过量（1 mM）的5-羟色胺或多巴胺时，结合并未减少。一旦形成，结合不会被螯合剂（如乙二醇四乙酸和去铁胺）破坏。其次，Fe(2+)刺激的结合会被还原剂（抗坏血酸钠、维生素E、焦亚硫酸钠）和消耗超氧自由基的试剂（超氧化物歧化酶和过氧化氢）抑制。此外，Fe2+的作用可以被氧化剂（高碘酸钠、超氧化钾）以及黄嘌呤氧化酶催化黄嘌呤氧化产生的超氧自由基模拟。为整合这些研究结果，我们提出一个假设：Fe2+与溶解的分子氧反应生成超氧自由基，这些自由基氧化[3H]5-羟色胺和[3H]多巴胺，并且形成的氧化产物与SBP的半胱氨酸残基共价结合。这种替代机制还基于含有或修饰巯基的试剂能够降低结合，以及羟基自由基清除剂（二甲基亚砜、甘露醇、乙醇和硫脲）不能降低结合的能力。Fe2+还能够不可逆地使SBP上的部分结合位点失活（[3H]5-羟色胺特异性结合的81%，[3H]多巴胺的61%）。这种Fe(2+)介导的失活以及结合的共价性质，使得无法根据可逆双分子相互作用来解释饱和结合和竞争结合数据。然而，此类实验表明，在相同浓度下，[3H]多巴胺结合的位点数量是[3H]5-羟色胺的2至3倍。未标记的多巴胺在所有[3H]5-羟色胺结合位点上也是一种有效的竞争者，而未标记的5-羟色胺仅在部分（30%）[3H]多巴胺结合位点上是一种有效的竞争者。SBP最初被认为参与5-羟色胺的储存、保护和/或运输，最近也被认为参与儿茶酚胺的相关过程。然而，SBP的这些潜在功能很难与结合的分子机制相协调。此外，可以想象这种结合实际上代表了神经退行性变的一种体外模型。
