McKenna S M, Davies K J
Institute for Toxicology, University of Southern California, Los Angeles 90033.
Biochem J. 1988 Sep 15;254(3):685-92. doi: 10.1042/bj2540685.
The 'respiratory burst' of phagocytes such as neutrophils generates superoxide which forms H2O2 by dismutation. H2O2 and Cl- ions serve as substrates for the enzyme myeloperoxidase to generate hypochlorous acid (HOCl). HOCl is thought to play an important role in bacterial killing, but its mechanism of action is not well characterized. Furthermore, although many studies in vitro have shown HOCl to be a damaging oxidant with little or no specificity (particularly at high concentrations), bacteria which have been ingested by phagocytes appear to experience a rapid and selective inhibition of cell division. Bacterial membrane disruption, protein degradation, and inhibition of protein synthesis, do not seem to occur in the early phases of phagocyte action. We have now found that low concentrations of HOCl exert a rapid and selective inhibition of bacterial growth and cell division, which can be blocked by taurine or amino acids. Only 20 microM-HOCl was required for 50% inhibition of bacterial growth (5 x 10(8) Escherichia coli/ml), and 50 microM-HOCl completely inhibited cell division (colony formation). These effects were apparent within 5 min of HOCl exposure, and were not reversed by extensive washings. DNA synthesis (incorporation of [3H]-thymidine) was significantly affected by even a 1 min exposure to 50 microM-HOCl, and decreased by as much as 96% after 5 min. In contrast, bacterial membrane disruption and extensive protein degradation/fragmentation (release of acid-soluble counts from [3H]leucine-labelled cells) were not observed at concentrations below 5 mM-HOCl. Protein synthesis (incorporation of [3H]leucine) was only inhibited by 10-30% following 5 min exposure to 50 microM-HOCl, although longer exposure produced more marked reductions (80% after 30 min). Neutrophils deficient in myeloperoxidase cannot convert H2O2 to HOCl, yet can kill bacteria. We have found that H2O2 is only 6% as effective as HOCl in inhibiting E. coli growth and cell division (0.34 mM-H2O2 required for 50% inhibition of colony formation), and taurine or amino acids do not block this effect. Our results are consistent with a rapid and selective inhibition of bacterial cell division by HOCl in phagocytes. H2O2 may substitute for HOCl in myeloperoxidase deficiency, but by a different mechanism and at a greater metabolic cost.
中性粒细胞等吞噬细胞的“呼吸爆发”会产生超氧化物，超氧化物通过歧化反应形成过氧化氢（H₂O₂）。H₂O₂和氯离子作为髓过氧化物酶的底物，生成次氯酸（HOCl）。HOCl被认为在杀灭细菌中起重要作用，但其作用机制尚未完全明确。此外，尽管许多体外研究表明HOCl是一种具有很少或没有特异性的破坏性氧化剂（特别是在高浓度时），但被吞噬细胞吞噬的细菌似乎会经历细胞分裂的快速且选择性抑制。在吞噬细胞作用的早期阶段，似乎并未发生细菌膜破坏、蛋白质降解以及蛋白质合成抑制。我们现在发现，低浓度的HOCl会对细菌生长和细胞分裂产生快速且选择性的抑制作用，这种作用可被牛磺酸或氨基酸阻断。抑制50%细菌生长（5×10⁸/ml大肠杆菌）仅需20μM的HOCl，50μM的HOCl可完全抑制细胞分裂（菌落形成）。这些效应在HOCl暴露5分钟内就很明显，且大量洗涤不能逆转。即使仅暴露于50μM的HOCl 1分钟，DNA合成（[³H]胸腺嘧啶核苷掺入）就会受到显著影响，5分钟后减少多达96%。相比之下，在低于5mM的HOCl浓度下未观察到细菌膜破坏和广泛的蛋白质降解/片段化（从[³H]亮氨酸标记的细胞中释放酸溶性计数）。暴露于50μM的HOCl 5分钟后，蛋白质合成（[³H]亮氨酸掺入）仅被抑制10% - 30%，尽管更长时间的暴露会产生更明显的减少（30分钟后为80%）。缺乏髓过氧化物酶的中性粒细胞无法将H₂O₂转化为HOCl，但仍能杀灭细菌。我们发现，在抑制大肠杆菌生长和细胞分裂方面，H₂O₂的效果仅为HOCl的6%（抑制50%菌落形成需要0.34mM的H₂O₂），且牛磺酸或氨基酸不能阻断这种作用。我们的结果与吞噬细胞中HOCl对细菌细胞分裂的快速且选择性抑制一致。在髓过氧化物酶缺乏时，H₂O₂可能替代HOCl，但通过不同机制且代谢成本更高。
