Distinction between nitrosating mechanisms within human cells and aqueous solution
Distinction between nitrosating mechanisms within human cells and aqueous solution
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DOI：
10.1074/jbc.m101723200
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发表时间：
2001-08-10
影响因子：
4.8
通讯作者：
Wink, DA
中科院分区：
文献类型：
--
作者：
Espey, MG;Miranda, KM;Wink, DA
在非自氧化过程中产生的典型亚硝化物种是N2O3。胺、硫醇和羟基残基的亚硝化可以调节关键的细胞功能。利用合成供体NaEt2NN(O)NO(DEA/NO)、人肿瘤细胞和4，5-二氨基荧光素(DAF)研究了在纳米到微摩尔水平的NO自氧化过程中形成的氮氧化物物种的反应活性的生物学机制。在好氧缓冲溶液中，DAF中NO的消失和亚硝化产物的生成都遵循二级过程，而完整细胞内的NO消耗和亚硝化反应是指数过程。以DEA/NO和2-苯基-4，4，5，5-四甲基咪唑-1-氧基-3-氧化物(PTIO)为最佳摩尔比，通过NO2的中间体生成N2O_3。这条途径被发现最能反映完整细胞内的亚硝化机制，不同于在水介质中发生的NO自氧化过程。对内源性清除剂抗坏血酸和谷胱甘肽的操纵表明，这些物质的位置、亲和力和浓度是决定分子靶标亚硝化敏感性的关键决定因素。综上所述，这些发现表明，亚硝化的功能效应可能被组织起来发生在离散的区域或隔室内。当清道夫耗尽时，亚硝化性压力可能会产生，这种结构会受到损害。尽管NO2不是水中NO自氧化的组成部分，但结果表明，这种物种的中间体可能是生物体系中亚硝化或氧化化学出现的一个重要因素。
The quintessential nitrosating species produced during NO autoxidation is N2O3. Nitrosation of amine, thiol, and hydroxyl residues can modulate critical cell functions. The biological mechanisms that control reactivity of nitrogen oxide species formed during autoxidation of nano- to micromolar levels of NO were examined using the synthetic donor NaEt2NN(O)NO (DEA/NO), hum an tumor cells, and 4,5-diaminofluorescein (DAF). Both the disappearance of NO and formation of nitrosated product from DAF in aerobic aqueous buffer followed second order processes; however, consumption of NO and nitrosation within intact cells were exponential. An optimal ratio of DEA/NO and 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) was used to form N2O3 through the intermediacy of NO2. This route was found to be most reflective of the nitrosative mechanism within intact cells and was distinct from the process that occurred during autoxidation of NO in aqueous media. Manipulation of the endogenous scavengers ascorbate and glutathione indicated that the location, affinity, and concentration of these substances were key determinants in dictating nitrosative susceptibility of molecular targets. Taken together, these findings suggest that the functional effects of nitrosation may be organized to occur within discrete domains or compartments. Nitrosative stress may develop when scavengers are depleted and this architecture becomes compromised. Although NO2 was not a component of aqueous NO autoxidation, the results suggest that the intermediacy of this species may be a significant factor in the advent of either nitrosation or oxidation chemistry in biological systems.