Carr A C, Winterbourn C C
Department of Pathology, Christchurch School of Medicine, P.O. Box 4345, Christchurch, New Zealand.
Biochem J. 1997 Oct 1;327 ( Pt 1)(Pt 1):275-81. doi: 10.1042/bj3270275.
Neutrophils, when stimulated, generate reactive oxygen species including myeloperoxidase-derived HOCl. There is an associated decrease in reduced glutathione (GSH) concentration. We have shown that neutrophil GSH levels decrease on exposure to reagent HOCl, whereas the equivalent concentration of H2O2 had no effect. GSH loss occurred without cell lysis, was not reversible, and was accompanied by the loss of an equivalent proportion of the total protein thiols. No glutathione disulphide was formed. Studies with 35S-labelled cells indicated that much of the GSH lost was accounted for by mixed disulphides with protein and a product that co-migrated on HPLC with a novel compound formed in the reaction of HOCl and pure GSH. The properties of this compound are consistent with an intramolecular sulphonamide. Neutrophils stimulated with PMA lost 30-40% of their GSH and a similar proportion of protein thiols. Little glutathione disulphide was formed and the products were the same as seen with HOCl-treated cells. From the results and studies with inhibitors and scavengers, we conclude that HOCl was responsible for the GSH loss. Propargylglycine and buthionine sulphoximine, inhibitors of glutathione synthesis, enhanced GSH loss, but their effects were due to the production of long-lived chloramines that oxidized GSH with greater efficiency than HOCl, rather than to the inhibition of GSH synthesis. The lack of thiol selectivity by HOCl and irreversibility of oxidation means that GSH will provide limited antioxidant protection for thiol enzymes in stimulated neutrophils.
中性粒细胞受到刺激时会产生活性氧，包括髓过氧化物酶衍生的次氯酸（HOCl）。同时，细胞内还原型谷胱甘肽（GSH）的浓度会相应降低。我们已经证明，中性粒细胞暴露于试剂HOCl时GSH水平会下降，而同等浓度的过氧化氢（H2O2）则没有这种作用。GSH的损失发生在细胞未裂解的情况下，是不可逆的，并且伴随着总蛋白巯基中相当比例的损失。没有形成谷胱甘肽二硫化物。用35S标记细胞的研究表明，损失的大部分GSH是由与蛋白质形成的混合二硫化物以及一种在HOCl与纯GSH反应中形成的、在高效液相色谱（HPLC）上共迁移的产物所导致的。该化合物的性质与分子内磺酰胺一致。用佛波酯（PMA）刺激的中性粒细胞损失了30 - 40%的GSH以及相似比例的蛋白巯基。几乎没有形成谷胱甘肽二硫化物，其产物与HOCl处理的细胞相同。根据这些结果以及使用抑制剂和清除剂的研究，我们得出结论，HOCl是导致GSH损失的原因。谷胱甘肽合成抑制剂炔丙基甘氨酸和丁硫氨酸亚砜胺会增强GSH的损失，但它们的作用是由于产生了寿命较长的氯胺，这些氯胺比HOCl更有效地氧化GSH，而不是因为抑制了GSH的合成。HOCl缺乏硫醇选择性以及氧化的不可逆性意味着GSH为受刺激的中性粒细胞中的硫醇酶提供的抗氧化保护有限。
