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 nature of iron released by resident and stimulated mouse peritoneal macrophages

Mouse peritoneal macrophages were allowed to ingest ⁵⁹Fe, ¹²⁵I-labelled transferrin-antitransferrin immune complexes, and the release of ⁵⁹Fe and degraded transferrin was studied. Some iron was released as ferritin, but a major portion was bound by bovine transferrin present in the culture medium, which contained fetal calf serum. If the medium was saturated with iron prior to incubation with the cells, little of the released iron was then bound by transferrin but appeared either as a high molecular weight fraction or, if nitrilotriacetate was present in the medium, some also appeared as a low molecular weight fraction. The release of non-ferritin iron was biphasic, the early, rapid phase being more prolonged with resident cells than with stimulated cells. The rate of release in the late phase did not differ significantly between resident and stimulated cells. Incubation at 0°C completely suppressed the release of degraded transferrin, but iron release continued at about 30% of the rate seen in control cultures at 37°C. A model for the intracellular handling of ingested iron is proposed to take account of the different release patterns of resident and stimulated macrophages.     

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 $\text{N-}\text{ethylmaleimide}$ 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°C but nearly all that taken up 4°C was released when the cells were subsequently incubated with trypsin plus $\text{N-}\text{ethylmaleimide}$, 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°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.     

The role of the anion-binding site of transferrin in its interaction with the reticulocyte

Specific and tight binding of Fe(III) by transferrin does not occur unless a suitable anion is concomitantly bound. Bicarbonate, which normally occupies the anion binding site of the protein, may be replaced by an oxalate ion. The resulting ternary complex of Fe(III), transferrin and oxalate is less than 35% as effective as the bicarbonate complex in serving as an iron donor for heme synthesis by the reticulocyte. However, the binding of transferrin to the reticulocyte is not altered by the substitution of oxalate for bicarbonate. When both the oxalate and bicarbonate forms are incubated with reticulocytes, the uptake of iron from the bicarbonate complex is substantially depressed. The free oxalate ion, at the same concentration as the ternary Fe-transferrin-oxalate complex, does not alter the uptake of iron by reticulocytes from the native form of transferrin. The ternary Fe-transferrin-malonate complex is also less efficient than the bicarbonate complex as an iron donor to the reticulocyte, but the effect is less striking than that observed with the oxalate complex. The hypothesis is advanced that the mechanism of iron uptake from transferrin during the transferrin-reticulocyte interaction first entails an attack upon the anion bound to the protein, following which iron release to the heme-synthesizing apparatus of the cell takes place.     

Correlation of proton release and ultraviolet difference spectra associated with metal binding by transferrin

A variety of metal ions can bind to the iron-transport protein, transferrin, at two specific sites. For each metal ion, a carboxylate anion is concomitantly bound. Six metal ions which were examined fall into two classes based on proton release and ultraviolet spectral changes which accompany binding to the protein. Class II ions, which include Cu²⁺ and Zn²⁺, release approximately 2 H⁺/metal bond. Class III ions, which include Fe³⁺, Ga³⁺, Al³⁺, and VO²⁺, release approximately 3 H⁺/metal bound. The increase in absorbance near 242 nm, characteristic of tyrosine ionization, has the ratio 0.55–0.75 for class II:class III ions. Both Fe³⁺ and Cu²⁺ form metal-transferrin-oxalate complexes in the presence of excess C₂O₄^2?. Fe³⁺ releases close to 3 H⁺/metal whether forming oxalate or bicarbonate complexes with transferrin. Binding of Cu²⁺ to transferrin releases 2 H⁺/metal in the presence of C₂O^2?₄ or HCO₃^?. Since equal numbers of H⁺/metal are released for both anions, it is likely that the bicarbonate ion does not lose its proton, and remains as HCO₃^? in transferrin. These results are interpreted in terms of possible combinations of ligands at the metal binding sites.     

Light versus Dark Carbon Metabolism in Cherry Tomato Fruits: II. Relationship Between Malate Metabolism and Photosynthetic Activity

The possible relationship between malate metabolism and photosynthetic activity in green tomato fruit tissues (Lycopersicum esculentum var. cerasiforme Dun A. Gray) was investigated. Initial experiments consisted of vacuum-infiltrating ¹⁴C-3 or ¹⁴C-4-malate into isolated tissues in darkness and then incubating the tissues under photosynthetic conditions. Other experiments involved a short pulse with ¹⁴C-bicarbonate in darkness to label the malate pool(s), followed by a chase in the light in the presence of nonradioactive bicarbonate. Both series of experiments were followed by the separation and identification of labeled metabolic intermediates.     

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.     

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 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.     

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.     

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.     

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.     

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.     

Iron release from transferrin induced by mixed ligand complexes of copper(II)

Summary Copper(II) complexes CuL₁L₂ with the ligand pairs 3-phosphoglycerate (PG)/ethylenediamine (en), phosphoserine (PS)/ethylenediamine, phosphoserine/malonate (mal) are shown to be effective in inducing the release of both iron atoms from di-ferric transferrin (Fe2Tf; human serum transferrin) at pH 7.3 in 1 M NaCl at 25°C. Half-times of the reaction with Cu(PG)(en)^– were less than 1 min at 0.02 M concentration. The iron(III) products are polynuclear hydroxo complexes. There is weaker interaction with Cu(PS) ₂ ^4– and virtually none with Cu(serine)(en) nor Cu(PS)(2,2-bipyridyl)^–, revealing crucial effects of the combined ligand sphere including the phosphomonoester group. The results suggest that the release of iron from Fe2Tf, or from either monoferric transferrins, occurred due to the breakdown of the stability of iron binding in conjunction with the expulsion of the synergistic anion carbonate (or oxalate). The active copper(II) complexes are postulated to be models of membrane components that could liberate iron from transferrin succeeding its uptake at the receptor sites of cells.Abbreviations PG phosphoglycerate - PS phosphoserine - en ethylenediamine - Fe2Tf diferric transferrin - Fe_cTf and Fe_NTf transferrin with iron bound to the lobe containing the C- or N-terminus, respectively - apoTf apotransferrin - K-3 all-cis-1,3,5-tris(trimethylammonio)-2,4,6-cyclo-hexanetriol - NTA nitrilotriacetic acid; bipy, 2,2-bipyridine; mal, malonate     

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.     

Substitution of carbonate by non-physiological synergistic anion modulates the stability and iron release kinetics of serum transferrin

Serum transferrin (sTf) is a bi-lobal protein. Each lobe of sTf binds one Fe³⁺ ion in the presence of a synergistic anion. Physiologically, carbonate is the main synergistic anion but other anions such as oxalate, malonate, glycolate, maleate, glycine, etc. can substitute for carbonate in vitro. The present work provides the possible pathways by which the substitution of carbonate with oxalate affects the structural, kinetic, thermodynamic, and functional properties of blood plasma sTf. Analysis of equilibrium experiments measuring iron release and structural unfolding of carbonate and oxalate bound diferric-sTf (Fe₂sTf) as a function of pH, urea concentration, and temperature reveal that the structural and iron-centers stability of Fe₂sTf increase by substitution of carbonate with oxalate. Analysis of isothermal titration calorimetry (ITC) scans showed that the affinity of Fe³⁺ with apo-sTf is enhanced by substituting carbonate with oxalate. Analysis of kinetic and thermodynamic parameters measured for the iron release from the carbonate and oxalate bound monoferric-N-lobe of sTf (Fe_NsTf) and Fe₂sTf at pH 7.4 and pH 5.6 reveals that the substitution of carbonate with oxalate inhibits/retards the iron release via increasing the enthalpic barriers.     

Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2

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 used of the weak acid 5,5-dimethyl-oxazolidine-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.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

The reticulocyte-mediated release of iron and bicarbonate from transferrin: Effect of metabolic inhibitors

The effect of three groups of metabolic inhibitors on the incorporation of Fe and release of bicarbonate from transferrin by rabbit reticulocytes was measured. Inhibitors which affect reticulocyte Fe and transferrin uptake to the same extent (sodium arsenite, N-ethylmaleimide and iodoacetamide); those which inhibit reticulocyte Fe uptake to a greater extent than transferrin uptake (NaN₃, NaF, NaCN, rotenone, oligomycin, 2,4-dinitrophenol and cycloheximide); and compounds which after reticulocyte heme synthesis (CoCl₂, isonicotinic acid hydrazide and hemin) were used. In each case the effect on Fe incorporation and bicarbonate release was the sameThus, additional evidence has been obtained for the idea that the reticulocyte-mediated release of Fe and bicarbonate from transferrin are tightly coupled. The results are consistent with the hypothesis that an enzymatic attack on transferrin-bound bicarbonate is involved in the removal of Fe from transferrin by erythroid cells.     

Utilization of lactoferrin-bound and transferrin-bound iron by Campylobacter jejuni

Campylobacter jejuni NCTC 11168 was capable of growth to levels comparable with FeSO₄ in defined iron-limited medium (minimal essential medium alpha [MEMα]) containing ferrilactoferrin, ferritransferrin, or ferri-ovotransferrin. Iron was internalized in a contact-dependent manner, with 94% of cell-associated radioactivity from either ⁵⁵Fe-loaded transferrin or lactoferrin associated with the soluble cell fraction. Partitioning the iron source away from bacteria significantly decreased cellular growth. Excess cold transferrin or lactoferrin in cultures containing ⁵⁵Fe-loaded transferrin or lactoferrin resulted in reduced levels of ⁵⁵Fe uptake. Growth of C. jejuni in the presence of ferri- and an excess of apoprotein reduced overall levels of growth. Following incubation of cells in the presence of ferrilactoferrin, lactoferrin became associated with the cell surface; binding levels were higher after growth under iron limitation. A strain carrying a mutation in the cj0178 gene from the iron uptake system Cj0173c-Cj0178 demonstrated significantly reduced growth promotion in the presence of ferrilactoferrin in MEMα compared to wild type but was not affected in the presence of heme. Moreover, this mutant acquired less ⁵⁵Fe than wild type when incubated with ⁵⁵Fe-loaded protein and bound less lactoferrin. Complementation restored the wild-type phenotype when cells were grown with ferrilactoferrin. A mutant in the ABC transporter system permease gene (cj0174c) showed a small but significant growth reduction. The cj0176c-cj0177 intergenic region contains two separate Fur-regulated iron-repressible promoters. This is the first demonstration that C. jejuni is capable of acquiring iron from members of the transferrin protein family, and our data indicate a role for Cj0178 in this process.