Morgan E H
Biochim Biophys Acta. 1981 Mar 20;642(1):119-34. doi: 10.1016/0005-2736(81)90143-7.
The aim of this investigation was to test the hypothesis that elevation of intracellular pH would inhibit iron uptake by reticulocytes. The experiments were performed with rabbit reticulocytes and iron bound to rabbit transferrin. Incubation of the cells with NH4Cl, (NH4)2CO3, CH3NH2 and (CH3)2NH was used in an attempt to increase intracellular pH. These substances were all found to inhibit iron uptake by reticulocytes. The mechanism of action of NH4Cl and CH3NH2 was investigated in detail. Similar results were found with both reagents. They inhibited iron uptake in a concentration-dependent manner, but produced a small increase in the cellular uptake of transferrin. The onset of action was rapid and the effect was reversible. There was no decrease in the number of transferrin-binding sites per cell and their apparent affinity for transferrin increased slightly, while the efficiency of iron removal from transferrin per binding site diminished greatly. The rate of transferrin release from reticulocytes was unaffected. NH4Cl did not affect the rate of iron release from transferrin in a cell-free system. Incubation of reticulocytes with 10 mM NH4Cl or CH3NH2 was found to produce an increase in intracellular pH of 0.05-0.15 pH units. The intracellular pH determined by use of the weak acid 5,5-dimethyloxazolidine-2,4-dione was significantly higher than that obtained with the weak base (CH3)2NH. By transmission electron microscopy it was shown that reticulocytes treated with NH4Cl or CH3NH2 have enlarged intracellular vesicles. The results are considered to support the hypothesis that iron release from transferrin in reticulocytes occurs as a result of protonation of the transferrin within intracellular vesicles. According to this hypothesis, weak bases such as NH3 and CH3NH2 inhibit iron release by neutralizing H+ within the vesicles.
本研究的目的是验证细胞内pH值升高会抑制网织红细胞摄取铁这一假说。实验采用兔网织红细胞和与兔转铁蛋白结合的铁进行。用氯化铵、碳酸铵、甲胺和二甲胺孵育细胞，试图提高细胞内pH值。结果发现，这些物质均能抑制网织红细胞摄取铁。对氯化铵和甲胺的作用机制进行了详细研究。两种试剂得到了相似的结果。它们以浓度依赖的方式抑制铁摄取，但使转铁蛋白的细胞摄取量略有增加。作用起效迅速且效果可逆。每个细胞中转铁蛋白结合位点的数量没有减少，它们对转铁蛋白的表观亲和力略有增加，而每个结合位点从转铁蛋白中去除铁的效率则大幅降低。网织红细胞中转铁蛋白的释放速率未受影响。在无细胞体系中，氯化铵不影响转铁蛋白中铁的释放速率。发现用10 mM氯化铵或甲胺孵育网织红细胞可使细胞内pH值升高0.05 - 0.15个pH单位。使用弱酸5,5 - 二甲基恶唑烷 - 2,4 - 二酮测定的细胞内pH值显著高于使用弱碱二甲胺所测得的值。通过透射电子显微镜观察显示，用氯化铵或甲胺处理的网织红细胞内的囊泡增大。这些结果被认为支持了以下假说：网织红细胞中转铁蛋白的铁释放是由于细胞内囊泡中转铁蛋白质子化所致。根据这一假说，诸如氨和甲胺之类的弱碱通过中和囊泡内的H⁺来抑制铁释放。
