Van Duijn M M, Van der Zee J, VanSteveninck J, Van den Broek P J
Department of Molecular Cell Biology, Sylvius Laboratory, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
J Biol Chem. 1998 May 29;273(22):13415-20. doi: 10.1074/jbc.273.22.13415.
The impermeable oxidant ferricyanide is reduced by the plasma membrane redox system of HL-60 cells. The rate of reduction is strongly enhanced by ascorbate or dehydroascorbate. The aim of this study was to determine the mechanism by which ascorbate and dehydroascorbate accelerate ferricyanide reduction in HL-60 cells. Addition of ascorbate or dehydroascorbate to cells in the presence of ferricyanide led to the intracellular accumulation of ascorbate. Control experiments showed that extracellular ascorbate was rapidly converted to dehydroascorbate in the presence of ferricyanide. These data suggest that intracellular ascorbate originates from extracellular dehydroascorbate. Accumulation of ascorbate was prevented by inhibitors of dehydroascorbate transport into the cell. These compounds also strongly inhibited ascorbate-stimulated ferricyanide reduction in HL-60 cells. Thus, it is concluded that the stimulation of ferricyanide reduction is dependent on intracellular accumulation of ascorbate. Changing the alpha-tocopherol content of the cells had no effect on the ascorbate-stimulated ferricyanide reduction, showing that a nonenzymatic redox system utilizing alpha-tocopherol was not involved. p-Chloromercuribenzenesulfonic acid strongly affected ferricyanide reduction in the absence of ascorbate, whereas the stimulated reaction was much less responsive to this compound. Thus, it appears that at least two different membrane redox systems are operative in HL-60 cells, both capable of reducing ferricyanide, but through different mechanisms. The first system is the ferricyanide reductase, which uses NADH as its source for electrons, whereas the novel system proposed in this paper relies on ascorbate.
不透性氧化剂铁氰化物可被HL-60细胞的质膜氧化还原系统还原。抗坏血酸盐或脱氢抗坏血酸盐可显著增强还原速率。本研究的目的是确定抗坏血酸盐和脱氢抗坏血酸盐加速HL-60细胞中铁氰化物还原的机制。在铁氰化物存在的情况下，向细胞中添加抗坏血酸盐或脱氢抗坏血酸盐会导致细胞内抗坏血酸盐的积累。对照实验表明，在铁氰化物存在的情况下，细胞外抗坏血酸盐会迅速转化为脱氢抗坏血酸盐。这些数据表明，细胞内抗坏血酸盐来源于细胞外脱氢抗坏血酸盐。脱氢抗坏血酸盐转运入细胞的抑制剂可阻止抗坏血酸盐的积累。这些化合物也强烈抑制HL-60细胞中抗坏血酸盐刺激的铁氰化物还原。因此，得出结论，铁氰化物还原的刺激依赖于细胞内抗坏血酸盐的积累。改变细胞中α-生育酚的含量对抗坏血酸盐刺激的铁氰化物还原没有影响，表明不涉及利用α-生育酚的非酶氧化还原系统。对氯汞苯磺酸在没有抗坏血酸盐的情况下强烈影响铁氰化物还原，而受刺激的反应对该化合物的反应性要小得多。因此，似乎至少有两种不同的膜氧化还原系统在HL-60细胞中起作用，两者都能够还原铁氰化物，但通过不同的机制。第一个系统是铁氰化物还原酶，它使用NADH作为电子来源，而本文提出的新系统依赖于抗坏血酸盐。
