Competitive advantage of diferric transferrin in delivering iron to reticulocytes.

Radioiron- and radioiodine-labeled forms of human diferric and monoferric transferrin and apotransferrin, isolated by preparative isoelectric focusing, were used to define transferrin-iron uptake by human reticulocytes. In mixtures of human diferric and monoferric transferrin, the diferric molecule had a constant 7-fold advantage in delivering iron to reticulocytes, as compared with the 2-fold advantage when single solutions of mono- and diferric transferrins were compared. This was shown to be due to competitive interaction in iron delivery, probably at a common membrane-receptor binding site for transferrin. Apotransferrin did not interfere with the iron-donating process and its limited cellular uptake was inhibited in noncompetitive fashion by diferric transferrin.     

Uptake and release of iron from human transferrin.

Purified fractions of human apotransferrin, monoferric transferrins with iron on the acid-labile binding site and on the acid-stable binding site, and diferric transferrin have been prepared. The iron loading and unloading behavior of these preparations has been examined by isoelectric focusing. Iron release from the two monoferric transferrin preparations to human reticulocytes was of similar magnitude. In a mixture containing equal amounts of diferic and monoferric iron, approximately 4 times the amount of iron delivered by the monoferric species was delivered by the diferric species. Iron loading of transferrin in vitro showed a random distribution between monoferric and diferric transferrin. Among the monoferric transferrins, loading of the acid-labile binding sites was greater than that of the acid-stable binding sites. In vivo iron distribution in normal subjects, as evaluated by in vitro-added 50Fe, gave similar results. Absorption of a large dose of orally administered iron in iron-deficient subjects resulted in a somewhat greater amount of diferric transferrin at low saturation and a somewhat smaller amount of diferric transferrin at higher saturations than would have been anticipated by random loading. These data would indicate that in the human, iron loading of transferrin may be considered essentially random. Unloading from the two monoferric transferrin species is of similar magnitude but far less than that delivered by diferric transferrin.     

Interaction of human diferric transferrin with reticulocytes.

Methods have been devised for preparing human transferrin with a different isotope of iron selectively labeling each of the two iron binding sites and for determining the distribution of radioiron among transferrin molecules. When diferric human transferrin was exposed to human or animal reticulocytes, there was an equal contribution of radioiron from the acid-stable and acid-labile sites. In this delivery, both atoms of iron were removed simultaneously from the diferric transferrin molecule, converting it to apotransferrin. At similar iron concentrations the amount of iron delivered by diferric transferrin was twice that delivered by monoferric transferrin.     

Molecular ferrokinetics in the rabbit

S ummary . Using urea-polyacrylamide gel electrophoresis it has been possible to distinguish the molecular forms of transferrin in rabbit serum. When ⁵⁹Fe-labelled diferric transferrin is injected into normal, anaemic or hypertransfused, polycythaemic rabbits, iron is removed from diferric transferrin in essentially pairwise fashion. Exchange of iron between transferrin and tissues was also studied using predominantly monoferric transferrin labelled with ⁵⁹Fe or ¹²⁵I, and with ¹²⁵I-labelled apotransferrin. The return of iron from tissue stores to circulating transferrin occurs one atom at a time to either site of the protein and, possibly, in pairwise fashion as well. The rate of clearance of iron from diferric transferrin differs from that of monoferric transferrins, and the rates at which iron is returned to empty sites of transferrin also differ, so that serum iron is not a kinetically homogeneous pool in the rabbit.     

A reversible defect of platelet PDGF content in myeloproliferative disorders

The transport of iron through erythroid cell membrane was studied in a model system, measuring ferrous iron uptake by reticulocytes. It was found that these cells were able to take up ferrous iron and to incorporate it into haem at a rate similar to that observed when diferric transferrin was the iron donor. No comparable iron uptake could be measured when the metal was provided as Fe3+-citrate or when reticulocytes were replaced by mature erythrocytes. The involvement of endogenous transferrin in the Fe2+ uptake by reticulocytes could be excluded, since proteolytic treatment of the cells had no significant effect on the process. Fe2+ uptake by reticulocytes followed saturation kinetics, characteristic to carrier mediated transport processes. Kinetic analysis of the data revealed the following apparent transport parameters: Km = 8.8 +/- 3.8 microM; Vmax = 1.1 +/- 0.2 ng/10(8) reticulocytes/min. These results indicate that a high affinity, carrier mediated iron transport system is present in the reticulocyte membrane, ensuring the efficient translocation of the metal through the membrane barrier between the site of its release from transferrin and the site of its utilization.     

Functional heterogeneity of transferrin-bound iron: iron uptake by cell suspensions from bone marrow and liver and by cell cultures of fibroblasts and lymphoblasts.

According to the hypothesis of Fletcher and Huehns, functional differences exist between both iron-binding sites of transferrin. The site designated A should mainly be involved in the delivery of iron to erythroid cells, whereas site B should donate its iron preferentially to cells involved in the absorption and storage of iron. In the present study this hypothesis could be confirmed by in vitro experiments with various cell types. Iron transferrin preincubated with rat bone marrow cells donates less iron to rat bone marrow cells, Chinese hamster fibroblasts, human fibroblasts and human lymphoblasts than freshly prepared iron transferrin equal in iron and transferrin concentraion. Rat liver parenchymal cells, however, take up more iron from preincubated than from freshly prepared iron transferrin. Obviously, site A not only donates iron preferentially to erythroid cells but also to (rapidly) dividing nonerythroid cells in culture. From experiments with iron transferrin mixtures in which radioiron was present at low or high iron saturation, it could be concluded that rat bone marrow cells take up iron equally well from monoferric as from diferric transferrin. The observed functional heterogeneity could, therefore, not be ascribed to differences between monoferric and diferric transferrin.     

Transferrin—Reticulocyte Binding: Evidence for the Functional Importance of Transferrin Conformation

Iron-free and ⁵⁹Fe-labelled human and rabbit ¹²⁵I-transferrins have been chromatographed on DEAE cellulose at pH 7.9 and have also been incubated with rabbit reticulocytes in order to determine transferrin and iron uptake.
Chromatography of human apo- and iron-transferrin indicated clear differences in their surface properties which are considered to be conformation-dependent. These differences were not evident in rabbit transferrin samples.
Human and rabbit iron-transferrins and rabbit apo-transferrin all associated rapidly with rabbit reticulocytes; human apo-transferrin was only weakly bound. Correlation of data from both experiments suggested that reticulocyte binding of human apo-transferrin is limited by its molecular confirmation. The incubation studies confirmed results of other workers and indicated that the data from these experiments with human and rabbit transferrin are not in conflict.
Compared with rabbit iron-transferrin, only half as much human iron-transferrin was bound to rabbit reticulocytes in a comparable experiment. Iron transfer from bound human and rabbit transferrin took place at equal rate. It is argued that fewer receptors on rabbit reticulocytes are available for human transferrin because, when bound, the size and shape of a molecule limits further binding of transferrin molecules at adjacent sites.     

Studies on the Exchange of Iron between Transferrin and Reticulocytes

I ron is carried in the blood plasma very firmly bound to its specific carrying protein, transferrin, and in this form may be utilized for haemoglobin synthesis by reticulocytes (Walsh, Thomas, Chow, Fluharty and Finch, 1949) as well as by erythropoietic tissue of bone marrow. Although many workers have studied the exchange of iron between transferrin and reticulocytes (Paoletti, Boiron, Tubiana, Truhaut and Bernard, 1958; Jandl, Inman, Simmons and Allen, 1959; Allen and Jandl, 1960; Najean, Ardaillou and Bernard, 1960; Schade and Woodworth, 1961), knowledge of this process is still far from complete. Furthermore the results of various workers differ in certain important respects. For instance Jandl and associates (1959) found that iron uptake by reticulocytes varied with changes in transferrin concentration, while Schade and Woodworth (1961) found it to be independent of transferrin concentration. Similarly it is uncertain whether iron taken up by reticulocytes can (Pollycove and Maqsood, 1962) or cannot (Jandl et al. , 1959) be eluted by subsequent incubation with transferrin solutions. The present investigation was undertaken to provide further information on the mechanism of iron transfer from transferrin to reticulocytes and in an attempt to settle some of the differences in the results of previous workers. Rabbit reticulocytes, and rabbit plasma and rabbit transferrin purified by very mild procedures were used.     

Iron uptake by human reticulocytes at physiologic and sub-physiologic concentrations of iron transferrin: the effect of interaction with aluminum transferrin

We have studied the interaction, in vitro, between diferric transferrin (FeTr), aluminum transferrin (AlTr), and human reticulocytes harvested from human placental blood. In particular, we aimed to determine the extent to which the kinetics of AlTr and FeTr differed. Using transferrin labeled with either 59Fe or 125I, the association of radiotracer with reticulocytes, as a function both of time and of transferrin concentration, was examined. Under the conditions of the experiments, the data are consistent with a mechanism involving at least three processes. Two early processes acting in parallel behave as a high-affinity saturable receptor and a low-affinity non-saturable receptor, neither of which distinguish between AlTr and FeTr. In a subsequent process, AlTr and FeTr exhibit different kinetics. This third process may be saturated by FeTr but not by AlTr. Interpreted in terms of a current conventional view of metallo-transferrin uptake, we conjecture that the early parallel processes involve cell surface phenomena including classical transferrin-receptor binding, and that the subsequent process represents events, possibly intracellular, involved in metallo-transferrin dissociation or further iron transport. The extent to which AlTr influences the interaction of FeTr with reticulocytes offers insight into both the normal physiology of iron uptake and the potential for toxicity by aluminum.     

Distribution of iron between the binding sites of transferrin in serum: methods and results in normal human subjects.

When it is incompletely saturated with iron, transferrin may exist in four molecular forms: apotransferrin, monoferric (A) transferrin (with iron occupying only the A site of the protein), monoferric (B) transferrin, and diferric transferrin. By combining electrophoresis in urea-polyacrylamide gels with crossed immunoelectrophoresis using specific antihuman transferin antiserum, it is possible to display and estimate the concentration of each of these four forms in normal human serum. The distribution of iron between the binding sites of transferrin is neither random nor determined by the relative binding strengths of transferrin's two sites. Rather, the more weakly binding and acid-labile B site of the protein is predominantly occupied.     

The behavior of transferrin iron in the rat

The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.     

Diminished acquisition of iron by reticulocytes from mice with hemoglobin deficit

Reticulocyte iron and transferrin uptake was studied in hemoglobin deficit (gene symbol, hbd), an autosomal recessive trait in the mouse characterized by hypochromic microcytic anemia, reticulocytosis, hyperferremia, and increased red-cell-free protoporphyrin. Reticulocyte-rich red cells were incubated in vitro in a mixture of 125I-labeled diferric mouse transferrin and 59Fe-labeled iron-saturated mouse plasma. At 37 degrees C, the uptake of transferrin by reticulocytes from affected animals (15 ng/micrograms RNA) was the same as that of reticulocytes from control animals. However, the uptake of iron by affected reticulocytes (0.11 ng/micrograms RNA) was significantly lower than that by control reticulocytes (0.24). At 4 degrees C, transferrin binding by affected and control reticulocytes was again indistinguishable. The deficiency in the uptake of iron by affected reticulocytes was not observed on incubation at 4 degrees C. Scatchard analysis of transferrin receptors on hbd/hbd and control reticulocytes showed no difference in pKD and a slight elevation in number of receptors per reticulocyte for hbd/hbd animals. These findings suggest that hbd/hbd reticulocytes have a defect in iron acquisition that is distal to the binding of transferrin to the cell membrane receptor. This defect is similar to one already described in the anemia of the Belgrade laboratory rat.     

Transferrin-reticulocyte cycle time in rat reticulocytes

Summary The uptake and release of¹³¹I-labelled diferric transferrin by rat reticulocytes was examined both in vitro and in vivo. Cycle time in vitro was estimated to be 2.5 min in iron-deficient reticulocytes and 2.3 min in phenylhydrazine-produced reticulocytes. In vivo reticulocyte uptake and release of labelled diferric transferrin injected in the iron-deficient rat averaged 1.7 min.     

Iron Uptake by Rabbit Reticulocytes

S ummary . In keeping with present concepts of internal iron exchange, it is thought that the two iron binding sites of transferrin molecules bind and donate iron in a similar manner. Since recent data, however, have suggested that iron attached at one iron binding site may be more readily available to cells than iron bound to the other site, a further study of in vitro iron exchange between transferrin and reticulocytes has been undertaken.
Transferrin samples were first incubated with reticulocytes to reduce the transferrin iron saturation; rates of iron uptake from incubated transferrin samples and appropriate non-incubated controls with the same iron saturation were then compared. At 24% transferrin iron saturation, iron uptake from incubated transferrin was significantly less than from the control; at 48% transferrin iron saturation, iron uptake was the same from incubated transferrin and controls.
Two transferrin samples of equal protein concentration were prepared; each was 50% iron saturated. One sample contained twice the proportion of iron saturated transferrin molecules (2Fe-transferrin) present in the other sample. Iron uptake by reticulocytes was decreased by 22–25% from the sample with less 2Fe-transferrin molecules. Thus iron was exchanged very rapidly between 2Fe-transferrin and reticulocytes.
It is proposed that the preliminary incubation of transferrin samples with reticulocytes caused a marked reduction in their 2Fe-transferrin molecules which, at low levels of transferrin iron saturation, resulted in the incubated samples containing less 2Fe-transferrin than the controls. In these circumstances, iron uptake from incubated transferrin was decreased when compared with the control.     

Transferrin and Iron Uptake by Rabbit Bone Marrow Cells in Vitro

S ummary . The interaction between rabbit transferrin and rabbit bone marrow cells and reticulocytes was investigated using transferrin labelled with ¹²⁵I and ⁵⁹Fe. The pattern of transferrin and iron uptake by bone marrow was found to be similar to that by reticulocytes and occurred in four stages, viz (1) adsorption of transferrin, (2) progressive uptake of transferrin, (3) release of the iron to the cell, (4) release of transferrin from the cell.
Total transferrin uptake per bone marrow erythroid precursor cell was eight times that per reticulocyte, while the rate of iron uptake was at least twice as great with marrow cells. Transferrin uptake and release and iron uptake were temperature dependent. Transferrin and iron uptake were inhibited by metabolic inhbitors, particularly those affecting oxidative metabolism. The activation energies for the association and dissociation reaction of transferrin and the activation energy for iron uptake by bone marrow cells were higher than those reported for reticulocytes. A further difference between the two cell types was that at a constant iron concentration, iron uptake by bone marrow cells decreased with decreasing degrees of transferrin saturation with iron. Small increases in transferrin uptake by bone marrow cells were also obtained with increasing transferrin saturation. The data support the view that both iron and transferrin uptake are dependent upon cellular metabolism.     

The Effects of Inhibitors of Microtubule and Microfilament Function on Transferrin and Iron Uptake by Rabbit Reticulocytes and Bone Marrow

S ummary . The possible involvement of microtubules and microfilaments in the uptake of transferrin and iron by rabbit reticulocytes and bone marrow cells was investigated by using agents which interfere with the functions of these cellular components. Colchicine, vinblastine, vincristine, strychnine, and heavy water, which inhibit microtubule function, all diminished transferrin and iron uptake by the cells. Vinblastine was also shown to inhibit transferrin release fiom reticulocytes and the uptake of both citrate-bound iron and leucine. Cellular ATP levels were not affected. Cytochalasin B which is believed to disrupt microfilaments did not affect transferrin or iron uptake by the cells. It is concluded that the inhibitory effects observed were due to alteration of structural components of the cell membranes, probably in the microtubules.     

Uptake of transferrin-bound and nontransferrin-bound iron by reticulocytes from the Belgrade laboratory rat: comparison with Wistar rat transferrin and reticulocytes.

The mechanism underlying the impaired uptake of iron from transferrin by reticulocytes from the Belgrade laboratory rat was investigated using 125I- and 59Fe-labeled transferrin isolated from homozygous Belgrade rats and from Wistar rats, nontransferrin-bound Fe(II) in an isotonic sucrose solution, and reticulocytes from Belgrade and Wistar rats. The Belgrade rat transferrin had the same molecular weight and net charge as Wistar rat transferrin, donated iron equally well to both types of reticulocytes, and competed equally for transferrin binding sites on the cells. Hence, the defect in iron uptake by Belgrade rat reticulocytes could not be attributed to an abnormality of the transferrin molecule. The rate of uptake of Fe(II) from sucrose into the cytosolic and stromal fractions of Belgrade rat reticulocytes was only about 35% as great as that by Wistar rat reticulocytes. With both types of cells, the uptake process was saturable, suggesting the presence of a carrier-mediated process. It was therefore concluded that the defect in iron uptake by Belgrade rat erythroid cells is probably the consequence of a deficiency in a membrane carrier for iron.     

Transferrin Glycans

The hepatic uptake of 59Fe from diferric rat and rabbit asialotransferrins and from human transferrin lacking two sialyl residues was investigated in rats in experiments lasting for 1 hr. The 59Fe attached to either of these preparations disappeared from the plasma more rapidly than the 59Fe introduced with the unmodified respective parent proteins. Most of the 59Fe activity that had disappeared from the circulation could be recovered with the liver. Studies with double-labeled (125I, 59Fe) preparations showed that the enhanced 59Fe clearance was not associated with increased catabolism of the modified transferrins. Prolonged, heavy alcohol consumption, as shown by others, results in the appearance of sialic acid-deficient transferrin (two residues missing) in human serum. We suggest that the increased capacity of transferrin deficient in sialic acid to selectively deposit iron in the hepatocyte may be of significance for the development of the hepatic siderosis observed in alcoholism.     

The Effect of pH upon Human Transferrin: Selective Labelling of the Two Iron-binding Sites

The influence of pH changes upon the iron-binding properties of transferrin was investigated in the absence of chelating agents. The effects were demonstrated by spectrophotometry, gel filtration, and by studies of the intermolecular transfer of 59Fe from transferrin to conalbumin. At pH values below 6.7, diferric transferrin readily loses iron. The monoferric molecule, which is relatively resistant to acid dissociation, is preferentially formed. A temporary reduction of pH provides a simple method for selectively attaching iron to one metal-binding site, and allows double isotopic labelling of the transferrin molecule. This technique may permit further investigation of the physiological properties of the two iron-binding sites.     

Plasma radioiron kinetics in man: explanation for the effect of plasma iron concentration.

The plasma iron turnover was measured in 19 normal subjects. A correlation was found between plasma iron concentration and plasma iron turnover. In addition to the turnover of 55Fe at normal plasma iron concentration (predominantly monoferric transferrin), a second turnover in which the labeled plasma was saturated with iron (to produce predominantly diferric transferrin) was studied with 50Fe. It was demonstrated that diferric transferrin had a greater rate of iron turnover but that the distribution between erythroid and non-erythroid tissues was unchanged. It was concluded that plasma iron turnover is dependent on the monoferric/diferric transferrin ratio in the plasma but that the internal distribution of iron is unaffected.