Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, USA.
Chem Res Toxicol. 2010 Feb 15;23(2):422-31. doi: 10.1021/tx900387k.
The reactivity of Zn(7)- and Cd(7)-metallothionein (MT) with S-nitrosopenicillamine (SNAP), S-nitrosoglutathione (GSNO), and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) was investigated to explore the hypothesis that metallothionein is a signficant site of cellular reaction of nitric oxide or NO compounds. Zn(7)-MT reacted with SNAP or GSNO only under aerobic conditions and in the presence of light, which stimulates the decomposition of S-nitrosothiolates to NO. Zn(2+) is released, and protein thiols are modified. DEA/NO, which degrades spontaneously to release NO, also reacted with Zn(7)-MT only when oxygen was present. Anaerobically, DEA/NO reacted with Zn(7)-MT in the presence of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, which converts NO to NO(2). Glutathione competed effectively with Zn(7)-MT for reactive nitrogen oxide species in reaction mixtures. Reaction of Cd(7)-MT with SNAP also required oxygen and light to react. In this case, only a fraction of the Cd(2+) bound to Cd(7)-MT was displaced by SNAP. Apo-metallothionein was much more reactive with SNAP and DEA-NO than Zn(7)- or Cd(7)-MT. TE671 and LLC-PK(1) cell lines were incubated with DEA/NO to examine the role that MT might play in the cellular reactions of this NO donor compound. Incubation of cells with 0-80 microM Zn(2+) for 24 h resulted in progressively increasing concentrations of Zn-unsaturated MT. One hour of cellular exposure to a range of DEA/NO concentrations followed by 24 h of incubation caused no evident acute toxicity at less than 0.45 mM. Preinduction of MT did not alter this response. The effects of DEA/NO on proteomic, metallothionein, and low molecular weight (LMW) thiol pools, including glutathione (GSH), were measured. Substantial fractions of the proteomic and LMW thiol pools underwent reaction with little dislocation of Zn(2+). In addition, one-third of the MT thiol pool reacted without labilizing any of the bound Zn(2+). These results demonstrated that it was free thiols associated with MT that reacted with DEA/NO not those bound to Zn(2+). Moreover, under the conditions of the experiments, DEA/NO reacted with the spectrum of cellular thiols in proportion to their fraction in the cytosol and did not preferentially react with MT sulfhydryl groups.
研究了 Zn(7)- 和 Cd(7)-金属硫蛋白 (MT) 与 S-亚硝基青霉胺 (SNAP)、S-亚硝基谷胱甘肽 (GSNO) 和 2-(N,N-二乙基氨基)-二氮烯-2-氧化物 (DEA/NO) 的反应性，以探讨金属硫蛋白是否是一氧化氮或 NO 化合物细胞反应的重要部位。Zn(7)-MT 仅在有氧条件下并在光照下与 SNAP 或 GSNO 反应，光照刺激 S-亚硝基硫醇分解为 NO。Zn(2+) 被释放，蛋白质巯基被修饰。自动降解释放 NO 的 DEA/NO 也仅在存在氧气时与 Zn(7)-MT 反应。在无氧条件下，存在 2-苯-4,4,5,5-四甲基咪唑啉-1-氧-3-氧化物时，DEA/NO 与 Zn(7)-MT 反应，该氧化物将 NO 转化为 NO(2)。谷胱甘肽在反应混合物中有效地与 Zn(7)-MT 竞争活性氮氧化物。SNAP 与 Cd(7)-MT 的反应也需要氧气和光照。在这种情况下，只有 SNAP 取代 Cd(7)-MT 结合的 Cd(2+) 的一部分。脱金属硫蛋白比 Zn(7)-或 Cd(7)-MT 与 SNAP 和 DEA-NO 反应更剧烈。用 DEA/NO 孵育 TE671 和 LLC-PK(1)细胞系，以研究 MT 可能在该 NO 供体化合物的细胞反应中所起的作用。用 0-80 μM Zn(2+)孵育细胞 24 小时导致 Zn-不饱和 MT 的浓度逐渐增加。细胞暴露于一系列 DEA/NO 浓度 1 小时，然后孵育 24 小时，在小于 0.45 mM 时不会造成明显的急性毒性。预先诱导 MT 不会改变这种反应。测量了 DEA/NO 对蛋白质组、金属硫蛋白和低分子量 (LMW) 巯基池（包括谷胱甘肽 (GSH)）的影响。蛋白质组和 LMW 巯基池的很大一部分发生反应，而 Zn(2+) 的位置没有明显改变。此外，三分之一的 MT 巯基池发生反应，而没有使任何结合的 Zn(2+)不稳定。这些结果表明与 DEA/NO 反应的是与 MT 相关的游离巯基，而不是与 Zn(2+)结合的巯基。此外，在实验条件下，DEA/NO 与细胞巯基的光谱反应与它们在细胞质中的分数成比例，而不是优先与 MT 巯基反应。
