Release of iron from the two iron-binding sites of transferrin by cultured human cells: modulation by methylamine.
We have investigated the effect of increasing concentrations of methylamine (5, 10, and 25 mM) on the removal of iron from the two iron-binding sites of transferrin during endocytosis by human erythroleukemia (K562) cells. The molecular forms of transferrin released from the cells were analyzed by polyacrylamide gel electrophoresis in 6 M urea. Endocytosis of diferric transferrin was efficient since greater than 10% of surface-bound protein escaped endocytosis and was released in the diferric form. Although transferrin exocytosed from control cells had been depleted of 80% of its iron and contained 65-70% apotransferrin, iron-bearing species were also released (15% C-terminal monoferric; 10% N-terminal; 10% diferric). The ratio of the two monoferric species (C/N) was 1.32 +/- 0.12 (mean +/- SD; n = 4), suggesting that iron in the N-terminal site was more accessible to cells. In the presence of methylamine there was a concentration-dependent increase in the proportion of diferric transferrin release (less than 80% at 25 mM) and a concomitant decrease in apotransferrin. Small amounts of the iron-depleted species, especially apotransferrin, appeared before diferric transferrin, suggesting that these were preferentially released from the cells. The discrepancy between the proportions of the monoferric transferrin species noted with control cells was enhanced at all concentrations of methylamine, most markedly at 10 mM when the C/N ratio was 2.4. The N-terminal site of transferrin loses its iron at a higher pH than the C-terminal site, and so by progressively perturbing the pH of the endocytic vesicle we have increased the difference between the two sites observed with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)
D001665:Binding Sites; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D007457:Iodine Radioisotopes; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D007951:Leukemia, Myeloid; D008744:Methylamines / Q000494:pharmacology*; D011485:Protein Binding; D014168:Transferrin / Q000378:metabolism*
D007457:Iodine Radioisotopes; D008744:Methylamines; D014168:Transferrin; C027451:methylamine; D007501:Iron