Preferential utilization in vitro of iron bound to diferric transferrin by rabbit reticulocytes.

59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.     

Functional heterogeneity and pH-dependent dissociation properties of human transferrin.

Human diferric transferrin was partially labeled with 59Fe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte 59Fe uptake experiments with chemically labeled preparations indicated that iron bound at near neutral pH was more readily incorporated by reticulocytes than iron bound at low pH. The pH-dependent iron dissociation studies of biologically labeled transferrin solutions indicated that Fe3+, bound at the site from which the metal was initially utilized by the cells, dissociated between pH 5.8 and 7.4. In contrast, lower pH (5.2--5.8) was required to effect dissociation of iron that has remained bound to the protein after incubation with reticulocytes. These findings suggest that each human transferrin iron-binding site has different acid-base iron-binding properties which could be related to the observed heterogenic rabbit reticulocyte iron-donating properties of human transferrin and identifies that the near neutral iron-binding site initially surrenders its iron to these cells.     

Functional heterogeneity and pH-dependent dissociation properties of human transferrin

Human diferric transferrin was partially labeled with ⁵⁹Fe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte ⁵⁹ uptake experiments with chemically labeled preparations indicated that iron bound at near neutral ph was more readily incorporated by reticulocytes than iron bound at low pH. The pH-dependent iron dissociation studies of biologically labeled transferrin solutions indicated that Fe³⁺, bound at the site from which the metal was initially utilized by the cells, dissociated between pH 5.8 and 7.4. In contrast, lower pH (5.2–5.8) was required to effect dissociation of iron that had remained bound to the protein after incubation with reticulocytes. These findings suggest that each human transferrin iron-binding site has different acid-base iron-binding properties which could be related to the observed heterogenic rabbit reticulocyte iron-binding properties of human transferrin and identifies that the near neutral iron-donating site initially surrenders its iron to these cells.     

Iron uptake studies on erythroid cells.

Iron uptake from 55Fe-labelled transferrin, ferric citrate and the two fungal sideramines, ferricrocin and fusigen was studied using four erythroid cell cultures: Friend virus-transformed erythroleukemic cells (mouse), transformed bone marrow cells, Detroit-98 (human), reticulocytes (bovine), bone marrow cells (rabbit). The present comparative study reveals pronounced differences in iron uptake behaviour. Compared to transferrin, ferric citrate and the sideramines are preferred in transformed erythroid cells. In reticulocytes transferrin and ferric citrate showed a better uptake as compared to the two sideramines. Primary bone marrow cells showed nearly equal iron uptake rates using transferrin or ferricrocin.     

Ferric-salicylaldehyde isonicotinoyl hydrazone, a synthetic iron chelate, alleviates defective iron utilization by reticulocytes of the Belgrade rat

The Belgrade rat has a hypochromic, microcytic anemia inherited as an autosomal recessive mutation. Although transferrin binds normally to reticulocytes and internalizes normally, iron accumulation into cells and heme is much slower than normal. We have investigated the role of the transferrin cycle in this mutant by bypassing transferrin iron delivery with the iron chelate ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH). Fe-SIH increases iron uptake into heme by Belgrade reticulocytes, restoring it almost to normal levels. This increase indicates that Fe-SIH delivers iron to a step in iron utilization that is after the Belgrade defect. Depleting reticulocytes of transferrin did not alter these observations. Failure to achieve above normal rates of iron incorporation could indicate damage due to chronic intracellular iron deficiency. Also, iron delivery by Fe-SIH restored globin synthesis to near-normal levels in Belgrade reticulocytes. The rates of glycine incorporation into porphyrin and heme in Belgrade reticulocytes incubated with Fe2-transferrin or Fe-SIH paralleled the rates of iron incorporation into heme. These data are consistent with the concept that iron availability limits protoporphyrin formation in rat reticulocytes. The protoporphyrin used for heme synthesis is provided by de novo synthesis and not by a pool of pre-existing protoporphyrin. The Belgrade defect occurs in the movement of iron from transferrin to a step prior to the ferrous state and insertion into heme. This defect diminishes the synthesis of heme and, consequently, that of protoporphyrin and globin.     

The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin.     

Iron uptake studies on erythroid cells

Iron uptake from ⁵⁵Fe-labelled transferrin, ferric citrate and the two fungal sideramines, ferricrocin and fusigen was studied using four erythroid cell cultures: Friend virus-transformed erythroleukemic cells (mouse), transformed bone marrow cells, Detroit-98 (human), reticulocytes (bovine), bone marrow cells (rabbit). The present comparative study reveals pronounced differences in iron uptake behaviour. Compared to transferrin, ferric citrate and the sideramines are preferred in transformed erythroid cells. In reticulocytes transferrin and ferric citrate showed a better uptake as compared to the two sideramines. Primary bone marrow cells showed nearly equal iron uptake rates using transferrin or ferricrocin.     

Failure of rabbit reticulocytes to incorporate conalbumin or lactoferrin iron.

Despite the remarkable molecular similarity of human lactoferrin and human transferrin, the results of this investigation indicate that human lactoferrin was unable to furnish rabbit reticulocytes with iron for heme synthesis. Although conalbumin closely resembles transferrin in many of its properties, conalbumin iron-binding differs from human transferrin iron-binding. There are conflicting reports in the literature regarding conalbumin's ability to furnish iron to reticulocytes. In this study, small amounts of lactoferrin or conalbumin were adsorbed to mature and immature cell surfaces but neither of these iron-binding proteins surrendered iron intracellularly to reticulocytes for heme synthesis.     

Iron transport from sepharose-bound transferrin

To ascertain whether transferrin need enter the reticulocyte to deliver its iron after the association of transferrin with the cell membrane, {¹²⁵I, ⁵⁹Fe-}labeled transferrin was covalently bound to Sepharose beads. Iron uptake from Sepharose-bound transferrin into rabbit reticulocytes was about 9% that from free transferrin while heme synthesis was more efficient at nearly 19%. Similar results were obtained with murine transferrin and murine reticulocytes.These results indicate that the entrance of transferrin inside the cell is not an obligatory step in the process of iron uptake in rabbit and murine reticulocytes.     

Iron and transferrin distribution in reticulocytes incubated with heme synthesis inhibitors

A high level of non-heme iron (either labelled or unlabelled) in mitochondria, ferritin and low-molecular-weight pool of reticulocytes was induced by preincubation with isonicotinic acid hydrazide or penicillamine together with either ⁵⁹Fe- or ⁵⁶Fe-labelled transferrin. Addition of apotransferrin during reincubation of ⁵⁹Fe-labelled reticulocytes was accompanied by the transfer of ⁵⁹Fe from low-molecular-weight pool to transferrin, which was found in the reticulocyte cytosol both free and bound to a carrier. Similarly, when cells were reincubated with ¹²⁵I-labelled transferrin, more ¹²⁵I-labelled radioactivity was found, in both free and carrier-bound transferrin peaks, in reticulocytes with a high level of low-molecular-weight cold iron than in control ones. These results suggest that transferrin enters reticulocytes takes up iron from low-molecular-weight pool.     

Transferrin and iron uptake by human lymphoblastoid and K-562 cells

Two human lymphoblastoid cell lines and K-562 cells were found to take up radioiodinated transferrin and transferrin-bound iron in amounts comparable to reticulocytes. These cell lines were also shown to possess transferrin receptors whose numbers and affinity for transferrin were similar to those of reticulocytes. However, unlike reticulocytes, in which at least 90% of the iron taken up is incorporated into heme, in the lymphoblastoid and K-562 cells only around 10% of the incorporated iron is found in heme. In addition, in contrast to the hemoglobin synthesizing cells, excess heme does not inhibit the removal of iron from transferrin by the lymphoblastoid and K-562 cells, suggesting that only during erythroid differentiation do cells acquire a specific mechanism for removing iron from transferrin which is subject to feedback inhibition by heme.     

Transferrin uptake and release by reticulocytes treated with proteolytic enzymes and neuraminidase.

The mechanism of transferrin uptake by reticulocytes was investigated using rabbit transferrin labelled with 125I and 59Fe and rabbit reticulocytes which had been treated with trypsin, Pronase or neuraminidase. Low concentrations of the proteolytic enzymes produced a small increase in transferrin and iron uptake by the cells. However, higher concentrations or incubation of the cells with the enzymes for longer periods caused a marked fall in transferrin and iron uptake. This fall was associated with a reduction in the proportion of cellular transferrin which was bound to a cell membrane component solubilized with the non-ionic detergent, Teric 12A9. The effect of trypsin and Pronase on transferrin release from the cells was investigated in the absence and in the presence of N-ethylmaleimide which inhibits the normal process of transferrin release. It was found that only a small proportion of transferrin which had been taken up by reticulocytes at 37 degrees C but nearly all that taken up 4 degrees C was released when the cells were subsequently incubated with trypsin plus N-ethylmaleimide, despite the fact that about 80% of the 59Fe in the cells was released in both instances. Neuraminidase produced no change in transferrin and iron uptake by the cells. These experiments provide evidence that transferrin uptake by reticulocytes requires interaction with a receptor which is protein in nature and that following uptake at 37 degrees C, most of the transferrin is located at a site unavailable to the action of proteolytic enzymes. The results support the hypothesis that transferrin enters reticulocytes by endocytosis.     

Amines as inhibitors of iron transport in rabbit reticulocytes

The effect of the known inhibitors of iron uptake, n-butylamine and NH4Cl, was examined at the molecular level to more precisely define the mechanisms by which these lysosomotropic agents block iron uptake by rabbit reticulocytes. Utilizing a rapid pulse-chase technique to follow the handling of a cohort of 59Fe, 125I-transferrin bound to rabbit reticulocytes, both amines were observed to have no effect on the cell-mediated release of 59Fe from internalized transferrin. The results indicated, however, that both agents acted to 1) retard the internalization of transferrin bound to transferrin receptors on the plasma membrane of reticulocytes, 2) retard the externalization of internalized transferrin, and 3) block the transport into the cytosol of iron released from transferrin.     

Transferrin receptors during rabbit reticulocyte maturation

Experiments were performed to examine the fate of transferrin receptors in reticulocytes as these cells mature in vivo to erythrocytes. Reticulocytosis, synchronized by administration of actinomycin D, was induced in adult rabbits. Simultaneous measurements were made of haematological parameters and the interaction between transferrin and reticulocytes while the cells matured in vivo to erythrocytes. As the reticulocytes matured there was a parallel decline in their ability to take up transferrin and transferrin iron. At the same time, there was a proportionate decrease in the density of receptors for transferrin on the reticulocyte surface. The affinity of the receptors for transferrin remained unaltered during the maturation process. It was concluded that the inability of erythrocytes to take up transferrin or its iron is due primarily to the loss of transferrin receptors from the maturing reticulocyte surface.     

Transferrin receptors during rabbit reticulocyte maturation.

Experiments were performed to examine the fate of transferrin receptors in reticulocytes as these cells mature in vivo to erythrocytes. Reticulocytosis, synchronized by administration of actinomycin D, was induced in adult rabbits. Simultaneous measurements were made of haematological parameters and the interaction between transferrin and reticulocytes while the cells matured in vivo to erythrocytes. As the reticulocytes matured there was a parallel decline in their ability to take up transferrin and transferrin iron. At the same time, there was a proportionate decrease in the density of receptors for transferrin on the reticulocyte surface. The affinity of the receptors for transferrin remained unaltered during the maturation process. It was concluded that the inability of erythrocytes to take up transferrin or its iron is due primarily to the loss of transferrin receptors from the maturing reticulocyte surface.     

Non-transferrin donors of iron for heme synthesis in immature erythroid cells

The mechanism of iron uptake from several iron-containing compounds by transferrin-depleted rabbit reticulocytes and mouse spleen erythroid cells was investigated. Iron complexes of DL-penicillamine, citrate and six different aroyl hydrazones may be utilized by immature erythroid cells for hemoglobin synthesis, although less efficiently than iron from transferrin. HTF-14, a monoclonal antibody against human transferrin, reacts with rabbit transferrin and inhibits iron uptake and heme synthesis by rabbit reticulocytes. HTF-14 had no significant effect on iron uptake and heme synthesis when non-transferrin donors of iron were examined. Ammonium chloride (NH4Cl) increases intracellular pH and blocks the release or utilization of iron from the internalized transferrin. NH4Cl only slightly affected iron incorporation and heme synthesis from non-transferrin donors of iron. Hemin inhibited transferrin iron uptake and heme synthesis, but had a much lesser effect on iron incorporation and heme synthesis from non-transferrin donors of iron. These results allow us to conclude that transferrin-depleted reticulocytes take up iron from all of the examined non-transferrin iron donors without the involvement of the transferrin/transferrin receptor pathway.     

The rate limiting step in the reticulocyte uptake of transferrin and transferrin iron. Effects of some incubation variables.

Reticulocytes incubated in an isotonic NaCl saline medium containing glucose, glutamine and amino acids, were able to detach both iron atoms from all the transferrin incorporated by them. In the absence of these metabolites, although transferrin uptake was the same, the reticulocytes failed to remove completely the iron from the transferrin which they incorporated. It has been shown before that there is unspecific as well as specific binding of transferrin to the reticulocyte. By incubating the cells in the presence of a high concentration of bovine serum albumin, we have been able to prevent the unsepcific attachment of transferrin. At least 94% of the iodinated transferrin was capable of donating its iron to the reticulocytes.     

Studies on the forms of iron-transferrin released from rabbit reticulocytes

Measurement of the distribution of the four species of transferrin, viz, apotransferrin, diferric transferrin and the two monoferric transferrin, before and after incubation of iron-rich rabbit transferrin with rabbit reticulocytes showed that not all transferrin released from the cells were in the form of apotransferrin. Instead, a mixture of all four species of the protein was released with apotransferrin and C-terminal monoferric transferrin being the major fractions. The buffer solution containing 125I-labelled transferrin showed a continuous gain in percentages in apotransferrin and C-terminal monoferric transferrin after each incubation with reticulocytes. The N-terminal monoferric transferrin, however, remained unchanged suggesting that in the process of transferrin uptake by cells, the diferric transferrin releases its iron from the acid-labile site at N-domain first before the other iron from the acid-stable site.     

Hepatocytes and reticulocytes have different mechanisms for the uptake of iron from transferrin

The uptake of iron from transferrin by isolated rat hepatocytes and rat reticulocytes has been compared. The results show the following. 1) Reticulocytes and hepatocytes express plasma membrane NADH:ferricyanide oxidoreductase activity. The activity, expressed per 10(6) cells, is approximately 60-fold higher in the hepatocyte than in the reticulocyte. 2) Hepatocyte plasma membrane NADH:ferricyanide oxidoreductase activity and uptake of iron from transferrin are stimulated by low oxygen concentration and inhibited by iodoacetate. In reticulocytes, similar changes are seen in NADH:ferricyanide oxidoreductase activity, but not on iron uptake. 3) Ferricyanide inhibits the uptake of iron from transferrin by hepatocytes, but has no effect on iron uptake by reticulocytes. 4) Perturbants of endocytosis and endosomal acidification have no inhibitory effect on hepatocyte iron uptake, but inhibit reticulocyte iron uptake. 5) Hydrophilic iron chelators effectively inhibit hepatocyte iron uptake, but have no effect on reticulocyte iron uptake. Hydrophobic iron chelators generally inhibit both hepatocyte and reticulocyte iron uptake. 6) Divalent metal cations with ionic radii similar to or less than the ferrous iron ion are effective inhibitors of hepatocyte iron uptake with no effect on reticulocyte iron uptake. The results are compatible with hepatocyte uptake of iron from transferrin by a reductive process at the cell surface and reticulocyte iron uptake by receptor-mediated endocytosis.     

An evaluation of lysosomal enzyme leakage as a factor influencing the behaviour of transformed cells.

The effect of concanavalin A on transferrin and iron uptake by reticulocytes was determined using rabbit reticulocytes and rabbit transferrin labelled with ⁵⁹Fe and ¹²⁵I and concanavalin A (ConA) labelled with ¹³¹I. In concentrations of 50–200 μg/ml ConA markedly inhibited iron uptake but did not inhibit transferrin uptake or release from the cells. ConA was itself taken up by rabbit blood cells in a manner similar to that of transferrin except that the uptake was not specific for reticulocytes but occurred also with mature erythrocytes. The inhibition of iron uptake by concanavalin and the uptake of concanavalin by the cells were both inhibited by α-methyl-d-mannoside. It is concluded that the effects observed were due to the binding of concanavalin to glycoproteins of the cell membrane, either by a direct interaction with transferrin receptors or by the production of a non-specific change in the structure of the membrane.