The effect of acid pH and citrate on the release and exchange of iron on rat transferrin.

The effect of acid pH and citrate on the exchange of iron between binding sites of rat transferrin has been studied. In the absence of citrate, diferric transferrin shows stepwise loss of iron atoms with the first atom of iron released at approximately pH 5.2. Citrate at physiologic concentrations (1.10(-3) M) or greater allows random iron removal at pH 6.5 or less. Iron dissociation from monoferric transferrin at acid pH, with or without citrate, is a random process. At pH 7.4, randomization of iron on transferrin takes from 3 to 6 h in the presence of millimolar concentrations of citrate. We conclude that at acid pH and in the presence of citrate concentrations likely to occur in vivo in the rat there is little scrambling of iron bound to transferrin.     

The preparation, properties, and metabolism of 14C-bicarbonate-labelled transferrin

¹⁴C-bicarbonate-labelled transferrin was prepared in order to study the role of bicarbonate in the cell-mediated release of iron from transferrin. ¹⁴C-bicarbonate bound to transferrin only in the presence of iron and with a ratio of bound bicarbonate to bound iron of one. The transferrin-¹⁴C-bicarbonate complex was very stable in Tris-HCl buffered at pH 7.5–9.0 even in the presence of excess non-radioactive bicarbonate. However, oxalate, citrate, and phosphate promoted a rapid exchange of transferrin-bound ¹⁴C-bicarbonate with bicarbonate present in the medium.Rabbit reticulocytes effected a temperature-dependent release of ¹⁴C-bicarbonate from transferrin at the same rate at which they incorporated ⁵⁹Fe from transferrin — suggesting the existence of a coordinated mechanism in the cells for the release of both iron and bicarbonate from transferrin.     

Comparative study of the iron-binding properties of human transferrins: I. Complete and sequential iron saturation and desaturation of the lactotransferrin

Human lactotransferrin binds 2 Fe³⁺ tightly at two specific sites. In order to demonstrate differences between the stability of the two iron-binding sites, the removal of iron was studied in buffers in the pH range 8-3 varying the ionic strength and with or without metal chelators such as phosphate ions and EDTA.The results show that in the presence of formate and acetate buffers of ionic strength 0.1–0.4 and in a pH range of 5–3, the two Fe³⁺ from human lactotransferrin are removed stimultaneously.Addition of 4 mM EDTA to buffers of ionic strength 0.1 and in the pH range 8–3 shows that between pH 5–4.3 the iron from only one of the binding sites, called the ‘acid labile’ site, is removed.Addition of 0.2 M phosphate ions to buffers of ionic strength 0.2 and in pH range 8–3 containing 4 mM EDTA shows that Fe³⁺ from the ‘acid labile’ site may be completely removed at pH 6. Removal of Fe³⁺ from the ‘acid stable’ site is obtained at pH 4.The differential behavior of the two iron binding sites was also shown by saturation experiments in the presence of citrate/bicarbonate buffers at different pH values. In a pH range 6.2–4.8, 50% saturation was obtained, but at pH 6.35 complete saturation was achieved. When saturation of partially saturated samples of human lactotransferrin was performed with ⁵⁹Fe it was demonstrated that in the pH range 6.2–4.8 iron is bound only to the ‘acid labile’ site.     

Iron exchange between transferrin molecules mediated by phosphate compounds and other cell metabolites

The ability of a large number of cellular metabolites to release iron from transferrin was investigated by measuring the rate at which they could mediate iron exchange between two types of transferrin. Rabbit transferrin labelled with ⁵⁹Fe was incubated with human apotransferrin in the presence of the metabolites. After varying periods of incubation the human transferrin was separated from the rabbit transferrin by immunoprecipitation.GTP, 2,3-diphosphoglycerate, ATP, ADP and citrate produced the most rapid exchange of iron between the two types of transferrin, but many other compounds showed some degree of activity. Iron exchange mediated by the organic phosphates had the characteristics of a single first-order reaction and was sensitive to changes of incubation temperature and pH. The activation energy for the exchange reaction was approx. 13 kcal/mol. The rate of iron exchange from the oxalate · iron · transferrin complex was much lower than from bicarbonate · iron · transferrin.It is concluded that several organic phosphates have the capacity of releasing iron from transferrin. These compounds may represent the means by which the iron is released during the process of cellular uptake.     

The interaction of transferin with isolated hepatocytes

Freshly isolated rat heptocytes display about 36 700 high-affinity sites to which deferric transferrin may bind with an apparent association constant of 1.62·10⁷ 1·mol^?1.Uptake of iron from diferric transferrin by hepatocytes is linear with time and is accelerated at increased differric transferrin concentrations.Apotransferrin is able to decrease net iron uptake by hepatocytes from diferric transferrin by a process not dependent on the apotransferrin concentrations used or on the rate at which the cells take up iron. Immunoprecipitation of the apotransferrin during these incubations indicates that iron is being released from the cells to apotransferrin at the same time as iron is being taken up from diferric transferrin. The simultaneous uptake and release of iron, and the insensitivity to apotransferrin concentration, suggest that the processes of iron uptake and release occur via separate mechanisms. The effect of apotransferrin on net retention of iron may be one way in which the in vivo distribution of iron between sites of storage and utilization is controlled.     

Humic acid and iron uptake by plants

Summary The behaviour of ferric EDTA and ferric citrate in nutrient solution and their interaction with humic acid was investigated at various hydrogen ion concentrations using the technique of membrane ultrafiltration to separate small iron species from high molecular weight products of hydrolysis and to estimate the binding of iron by humic acid. Ferric EDTA was found to be of small molecular size at all pH values between 5.0 and 7.0 whilst ferric citrate solutions contained an increasing proportion of high molecular weight material as pH was increased from 5.0 to 7.0. Some iron present in solutions of both ferric EDTA and ferric citrate was bound by humic acid at all pH values from 5.0 to 7.0. Studies were also made of the uptake of iron by wheat roots from nutrient solutions containing either ferric EDTA or ferric citrate and of the effect of humic acid on uptake. More iron was absorbed from ferric EDTA than from ferric citrate at all pH values. Increasing pH between 5.0 and 7.0 resulted in a progressive decrease in the uptake of iron in both cases. The presence of humic acid depressed iron absorption from both solutions at all pH values.     

The activation of rabbit muscle, liver, and kidney fructose bisphosphatases by histidine and citrate

Purified fructose 1,6-bisphosphatases from rabbit muscle, liver, and kidney require a metal chelator for optimal activity at neutral pH. This requirement is satisfied by physiological concentrations of histidine and citrate, and at pH 7 their effects are additive. In the presence of both histidine and citrate the optimum activity is shifted from about pH 8 to pH 7.2, and the activity is greater than that obtained with optimal concentrations of EDTA. Carnosine, anserine, and 1-methyl histidine are also effective, but only at much higher concentrations, while 3-methyl histidine is effective in the same concentration range as is histidine. Isocitrate can replace citrate. The results suggest that fructose bisphosphatases possess distinct binding sites for divalent cations (Mg²⁺ or Mn²⁺) and also for histidine and citrate complexes of these cations.     

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.     

Growth of human tumor cell lines in transferrin-free,low-iron medium

Summary Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 μg/ml) or a higher concentration of ferric citrate (500 μM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 μM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 μM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.     

Effect of hemin and isonicotinic acid hydrazide on the uptake of iron from transferrin by isolated rat liver mitochonodria

Isolated rat liver mitochondria accumulate iron from the suspending medium when [⁵⁹Fe] transferrin is used as a model compound. The accumulation proceeds by two different mechanisms, i.e. by an energy-dependent and an energy-independent mechanism. The energy-dependent uptake of iron from transferrin is inhibited by hemin and stimulated by isonicotinic acid hydrazide. The energy-independent uptake of [⁵⁹Fe] transferrin is influenced neither by hemin nor by isonicotinic acid hydrazide.     

Studies of the binding of different iron donors to human serum transferrin and isolation of iron-binding fragments from the N- and C-terminal regions of the protein.

1. Trypsin digestion of human serum transferrin partially saturated with iron(III)-nitrilotriacetate at pH 5.5 or pH 8.5 produces a carbohydrate-containing iron-binding fragment of mol.wt. 43000. 2. When iron(III) citrate, FeCl3, iron (III) ascorabate and (NH4)2SO4,FeSO4 are used as iron donors to saturate the protein partially, at pH8.5, proteolytic digestion yields a fragment of mol.wt. 36000 that lacks carbohydrate. 3. The two fragments differ in their antigenic structures, amino acid compositions and peptide 'maps'. 4. The fragment with mol.wt. 36000 was assigned to the N-terminal region of the protein and the other to the C-terminal region. 5. The distribution of iron in human serum transferrin partially saturated with various iron donors was examined by electrophoresis in urea/polyacrylamide gels and the two possible monoferric forms were unequivocally identified. 6. The site designated A on human serum transferrin [Harris (1977) Biochemistry 16, 560--564] was assigned to the C-terminal region of the protein and the B site to the N-terminal region. 7. The distribution of iron on transferrin in human plasma was determined.     

Effect of pH on the transport of Krebs cycle intermediates in renal brush border membranes

Lowering extravesicular pH stimulated Na⁺-dependent citrate transport in renal brush border membrane vesicles: e.g., at pH_out = 5.5, the initial rate of citrate uptake was increased 10-fold compared to parallel control experiments at pH 7.5. The same experimental conditions had little effect on succinate uptake. The influence of pH on citrate transport is a product of the extravesicular H⁺ concentration; pH gradients did not potentiate the effects nor were proton gradients capable of driving transport in the absence of Na⁺. The effect of pH is adequately explained if only the mono- and divalent species of citrate (Cit^1?, Cit^2?) are considered acceptable substrates for transport. The stimulatory influence of pH on transport correlated quite well with pH-related increases in the concentrations of Cit^1? and Cit^2?, and over the same pH range [Cit^3?] was inversely related to citrate uptake. A model of the Na⁺-dependent dicarboxylate transport system is discussed in which three sodium ions are translocated per molecule of dicarboxylic acid.     

The effect of lysosomotrophic bases and inhibitors of transglutaminase on iron uptake by immature erythroid cells

The mechanism by which weak bases block iron uptake by immature erythroid cells was investigated using rabbit and rat reticulocytes and erythroblasts from the fetal rat liver. A large variety of bases was found to inhibit iron uptake but to have a much smaller or no effect on transferrin uptake by the cells. Quinacrine and chloroquine were active at the lowest concentrations. Dansylcadaverine, an inhibitor of transglutaminase, was also active at low concentration. However, the results do not indicate a role for transglutaminase in the iron uptake process. Instead they show that the major effect of the bases is to inhibit iron release from transferrin molecules on or within the cells. The possible mechanism of this effect was investigated by measurement of intracellular ATP levels, intracellular pH and by morphological studies utilizing fluorescent and electron microscopy. The bases caused little change in ATP levels, but elevated intracellular pH, probably due to accumulation within intracellular vesicles, which were shown to accumulate fluorescent weak bases, to swell under the action of the bases and to be the site of intracellular localization of transferrin. It is concluded that the bases tested in this work inhibit iron release from transferrin in intracellular vesicles by increasing their pH rather than by blocking transglutaminase and thereby restricting endocytosis. Reduction of transferrin uptake by the cells when it occurs is probably due to inhibition of recycling of transferrin receptors to the outer cell membrane.     

Effect of pH and oxalic acid on the reduction of Fe3+ by a biomimetic chelator and on Fe3+ desorption/adsorption onto wood: Implications for brown-rot decay

Fenton reaction is thought to play an important role in wood degradation by brown-rot fungi. In this context, the effect of oxalic acid and pH on iron reduction by a biomimetic fungal chelator and on the adsorption/desorption of iron to/from wood was investigated. The results presented in this work indicate that at pH 2.0 and 4.5 and in the presence of oxalic acid, the phenolate chelator 2,3-dihydroxybenzoic acid (2,3-DHBA) is capable of reducing ferric iron only when the iron is complexed with oxalate to form Fe³⁺-mono-oxalate (Fe(C₂O₄)⁺). Within the pH range tested in this work, this complex formation occurs when the oxalate:Fe³⁺ molar ratio is less than 20 (pH 2.0) or less than 10 (pH 4.5). When aqueous ferric iron was passed through a column packed with milled red spruce (Picea rubens) wood equilibrated at pH 2.0 and 4.5, it was observed that ferric iron binds to wood at pH 4.5 but not at pH 2.0, and the bound iron could then be released by application of oxalic acid at pH 4.5. The release of bound iron was dependent on the amount of oxalic acid applied in the column. When the amount of oxalate was at least 20-fold greater than the amount of iron bound to the wood, all bound iron was released. When Fe–oxalate complexes were applied to the milled wood column equilibrated in the pH range of 2–4.5, iron from Fe–oxalate complexes was bound to the wood only when the pH was 3.6 or higher and the oxalate:Fe³⁺ molar ratio was less than 10. When 2,3-DHBA was evaluated for its ability to release iron bound to the milled wood, it was found that 2,3-DHBA possessed a greater affinity for ferric iron than the wood as 2,3-DHBA was capable of releasing the ferric iron bound to the wood in the pH range 3.6–5.5. These results further the understanding of the mechanisms employed by brown-rot fungi in wood biodegradation processes.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

Studies on the mechanism of transferrin iron uptake by rat reticulocytes

Mechanism of transferrin iron uptake by rat reticulocytes was studied using ⁵⁹Fe- and ¹²⁵I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound ⁵⁹Fe was located in the cytoplasmic fraction, only 25–30% of ¹²⁵I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10–15% of the cytoplasmic ¹²⁵I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound ¹²⁵I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering ⁵⁹Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.     

An in vitro study on the binding of Al(III) to human serum transferrin with the isoelectric focusing technique

Transferrin saturated with Al³⁺ subjected to isoelectric focusing (IEF) in a pH gradient can be separated into four fractions, representing the apotransferrin, transferrin with aluminum at the metal binding site in the C- or N-terminal lobe, or both. The electrophoretic mobilities of these four fractions are identical to those of the iron-transferrin counterparts. Simultaneous binding of aluminum and iron to transferrin can also be demonstrated. The decreased saturation after IEF indicates that the affinity of transferrin for aluminum is low compared with its affinity for iron. This effect is particularly evident when bicarbonate is used as the synergistic anion in the loading procedure. In contrast, loading of transferrin with aluminum in the presence of oxalate produces a di-aluminum-transferrin complex that is stable during IEF.     

The effect of heterologous transferrin on the uptake of iron and haem synthesis by bone marrow cells

The initial process of transfer of extracellular iron to the haem-synthesizing mitochondria of immature erythroid cells is the association of iron-transferrin with the cell membrane. When rat bone marrow cells were incubated in the presence of iron bound to rat transferrin, iron uptake was higher than in the presence of iron bound to heterologous transferrin. The relative activities of the various isolated transferrins towards rat transferrin were found to be approximately 0.3, 0.8, 0.1 and 0.04 for rabbit, human, bovine and fish (tench, Tinca tinca) transferrin, respectively, and 0.7, 0.7 and 0.15 for mouse, guinea pig and calf serum, respectively, as compared with rat serum. Although great difference exist in cellular uptake of iron bound to different species of transferrin, the subcellular distribution of ⁵⁹Fe was quite similar. In all cases about 60% of the radioactivity taken up by the cells could be recovered in the haemin fraction and only about 15% in each the membrane and the non-haem soluble cell fraction. Similar results were obtained with guinea pig bone marrow cells.From the results of the experiments presented it might be concluded that the species of transferrin plays an important role during the initial stages of iron uptake by bone marrow cells, whereas the intracellular iron transfer process is not influenced by the species of transferrin.     

Molecular mechanisms of non‐transferrin‐bound and transferring‐bound iron uptake in primary hippocampal neurons

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell‐derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co‐localize, indicating these two proteins may function in Fe³⁺ reduction prior to Fe²⁺ permeation. Zip8, DMT1, and Steap2 co‐localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin‐mediated iron assimilation. In brain interstitial fluid, transferring‐bound iron (TBI) and non‐transferrin‐bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin‐⁵⁹Fe³⁺) and NTBI, whether presented as ⁵⁹Fe²⁺‐citrate or ⁵⁹Fe³⁺‐citrate; reductase‐independent ⁵⁹Fe²⁺ uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn²⁺ inhibition of Fe²⁺ uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of ⁵⁹Fe from TBI relies at least in part on an endocytosis‐independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons.