Transferrin Molecules Research Articles

Role of transferrin in iron transport between maternal and fetal circulations of a perfused lobule of human placenta.

The transfer of iron between the maternal and fetal circulations of an isolated perfused lobule of term human placenta was investigated using 125I-labelled or 59Fe-labelled diferric transferrin. There was negligible transplacental transfer of intact transferrin whereas nearly 4 per cent of the added 59Fe was transferred into the fetal circulation after 2 h, where it became associated with fetal transferrin. Over 20 per cent of the added 59Fe radioactivity was sequestered within the placental tissue during this period, associated with transferrin, ferritin and other uncharacterized molecules. This suggests an important role for an intracellular pool in regulating transfer. The presence of 10 mM chloroquine in the maternal circulation substantially reduced tissue accumulation of 59Fe and totally inhibited transfer to the fetus. It is concluded that the initial stages of iron transfer to the fetus involve the internalization of maternal iron-saturated transferrin bound to membrane receptors by receptor-mediated endocytosis, which can be inhibited by the drug chloroquine. Subsequently, the transplacental transfer of iron to the fetus does not involve the concomitant movement of transferrin.

Transferrin saturation, plasma iron turnover, and transferrin uptake in normal humans

The relationship between plasma iron, transferrin saturation, and plasma iron turnover was studied in 53 normal subjects whose transferrin saturation varied between 17% and 57%, in 25 normal subjects whose transferrin saturation was increased by iron infusion to between 67% and 100%, and in five subjects with early untreated idiopathic hemochromatosis whose transferrin saturation was continually elevated to between 61% and 86%. The plasma iron turnover of all of these subjects ranged from 0.45 to 1.22 mg/dL whole blood/d. The mean values for the above-mentioned three groups were 0.71 +/- 0.17, 1.01 +/- 0.11, and 1.01 +/- 0.13 mg/dL whole blood/d, respectively. Most of this variation, estimated at 72% by regression analysis, was due to a direct relationship between transferrin saturation and plasma iron turnover. This effect was attributed to a competitive advantage of diferric over monoferric transferrin in delivering iron to tissues. This was confirmed by the demonstration of a more rapid clearance of diferric as compared to monoferric transferrin in an additional group of eight normal subjects. Calculations were made of the amount of transferrin reacting with membrane receptors per unit time. Allowance was made for the noncellular (extravascular) exchange and for the 4.2:1 preference of diferric over monoferric transferrin demonstrated in vitro. The amount of iron-bearing transferrin leaving the plasma to bind to tissue receptors for 53 subjects with a transferrin saturation between 17% and 57% was 71 +/- 13; for 25 subjects with a saturation from 67% to 100%, 72 +/- 12; and for five subjects with early idiopathic hemochromatosis, 82 +/- 11 mumol/L whole blood/d. There were no significant differences among these groups. These studies indicate that while the number of iron atoms delivered to the tissues increases with increasing plasma iron and transferrin saturation, the number of iron-bearing transferrin molecules that leave the plasma per unit time to bind to tissue receptors is relatively constant and within the limits studied, independent of transferrin saturation.

Transferrin Saturation, Plasma Iron Turnover, and Transferrin Uptake in Normal Humans

The relationship between plasma iron, transferrin saturation, and plasma iron turnover was studied in 53 normal subjects whose transferrin saturation varied between 17% and 57%, in 25 normal subjects whose transferrin saturation was increased by iron infusion to between 67% and 100%, and in five subjects with early untreated idiopathic hemochromatosis whose transferrin saturation was continually elevated to between 61% and 86%. The plasma iron turnover of all of these subjects ranged from 0.45 to 1.22 mg/dL whole blood/d. The mean values for the above-mentioned three groups were 0.71 +/- 0.17, 1.01 +/- 0.11, and 1.01 +/- 0.13 mg/dL whole blood/d, respectively. Most of this variation, estimated at 72% by regression analysis, was due to a direct relationship between transferrin saturation and plasma iron turnover. This effect was attributed to a competitive advantage of diferric over monoferric transferrin in delivering iron to tissues. This was confirmed by the demonstration of a more rapid clearance of diferric as compared to monoferric transferrin in an additional group of eight normal subjects. Calculations were made of the amount of transferrin reacting with membrane receptors per unit time. Allowance was made for the noncellular (extravascular) exchange and for the 4.2:1 preference of diferric over monoferric transferrin demonstrated in vitro. The amount of iron-bearing transferrin leaving the plasma to bind to tissue receptors for 53 subjects with a transferrin saturation between 17% and 57% was 71 +/- 13; for 25 subjects with a saturation from 67% to 100%, 72 +/- 12; and for five subjects with early idiopathic hemochromatosis, 82 +/- 11 mumol/L whole blood/d. There were no significant differences among these groups. These studies indicate that while the number of iron atoms delivered to the tissues increases with increasing plasma iron and transferrin saturation, the number of iron-bearing transferrin molecules that leave the plasma per unit time to bind to tissue receptors is relatively constant and within the limits studied, independent of transferrin saturation.

The Identification of a Mathematical Model for the Investigation of the Differences Between Monoferric and Diferric Transferrin in Man

Abstract The internal iron exchange is mediated by transferrin, a carrier protein which has two iron binding sites. Recent studies, both “in vitro” and in animals, showed that a transferrin molecule bearing two iron atoms (diferric transferrin) delivers its iron to the tissues faster than a molecule bearing one iron atom (monoferric transferrin). We investigated the same phenomenon in man by using radioiron-labelled transferrin species. After having formulated a set of mathematical models able to describe the process, we studied the “a priori” identiftability of the models and designed the optimal experiment for parameters estimation. It consisted in the injection of two differently labelled samples of transferring one partially saturated with radioiron (a mixture of mono and diferrictransferrin) and the other completely saturated (diferric transferrin). The results obtained in normal subjects and patients with disorders of iron metabolism and erythropoiesis seem to indicate new interesting mechanisms of regulation of the iron transport from transferrin to tissues.

Transferrin and iron uptake by rat reticulocytes.

The uptake of transferrin labeled with 3H and 59Fe by rat reticulocytes was studied to clarify the characteristics of the uptake process and intracellular transport. Rat reticulocytes took up transferrin in a saturable, time- and temperature-dependent manner. Scatchard analysis of the binding parameters indicated that transferrin molecules were bound to cell-surface receptors with high affinity. Monodansyl- cadaverine, a potent inhibitor of transglutaminase, reduced the amount of internalized transferrin but has no effect on the total amount of cell-associated transferrin, suggesting that transferrin is taken up by rat reticulocytes via receptor-mediated endocytosis. About 50% of the internalized 3H label was released from the cells after reincubation for 1 h in fresh medium. In contrast, no release of 59Fe label was observed. By immunoprecipitation and subsequent SDS-PAGE the released 3H-labeled product was identified as apotransferrin. Lysosomotropic reagents and a proton ionophore reduced the uptake of 59Fe. These results indicated that iron was removed from transferrin at an intracellular site in an acidic environment. The released iron was found not to associate with any intermediate ligands before it was utilized for heme synthesis in mitochondria.

Phylogenetically more conservative epitopes among monoclonal antibody-defined antigenic sites of human transferrin are involved in receptor binding.

Of eight monoclonal antibodies raised against human transferrin, one (H.TF-14) cross reacted with pig and rabbit transferrins and one (H.TF-1) showed cross-reactivity with horse and dog transferrins. While rabbit and pig transferrins exhibited the same patterns of binding to MOLT-3 cell receptors as human and horse transferrins, binding of mouse and dog transferrins was weaker and bovine and carp transferrins gave entirely negative results. The results of these competitive binding experiments were confirmed by a biological test in which bovine transferrin had no effect on the growth of MOLT-3 cells when added to a serum-free medium. The observed correlation between cross-reactivity of anti-transferrin monoclonal antibodies and the binding abilities of transferrins to the MOLT-3 cell receptors may be associated with the conservatism of the part of the transferrin molecule recognized by the cell receptor.

Primate ( Macaca fascicularis) transferrin: Isolation and partial characterization

1. 1. The serum transferrin from the primate, Macaca fascicularis is isolated by a purification protocol consisting of ammonium sulphate precipitation and column chromatography. 2. 2. The hexose (galactose + mannose) content of Macaca transferrin is 4.7 mole per mole of protein. 3. 3. Quantitative determination of the sialic acid content shows that there are two sialic acid residues per molecule of Macaca transferrin. This conclusion is supported by the neuraminidase treatment of Macaca transferrin, in which there is a 2-step decrease in electrophoretic mobility. 4. 4. Monoferric Macaca transferrins with Fe 3+ selectively labelled at the C- and N-terminal sites (TfFe c and Fe NTf) are prepared at pH 5.5 and 8.5 using ferric dinitrilotriacetate [Fe(NTA) 2] chelate and ferrous ammonium sulphate, respectively.

A mono-sited transferrin from a representative deuterostome: the ascidian Pyura stolonifera (subphylum Urochordata)

An iron-binding protein has been found in the plasma of Pyura stolonifera. This protein has a molecular weight of about 41,000 +/- 2,000 and binds 1 mol iron/mol protein. The absorption maxima are lambda = 280 and lambda = 429 nm (E429/E280 = 0.044). Bicarbonate is bound concomitantly with high affinity and is necessary for optimal color formation at lambda = 429 nm. The protein showed a negligible exchange of iron with human apotransferrin under physiologic conditions over two hours. Upon incubation with rat reticulocytes, the protein reacts with membrane receptors for transferrins, and the protein, with its iron, is transported intracellularly where the iron is incorporated into heme. The 59Fe protein, after intravenous injection, disappears rapidly from the plasma and is excreted largely in the urine, with a substantial fraction present in the kidney and another large fraction present in the gut. These findings established the protein as a “transferrin” and support the concept that the larger transferrin molecule in vertebrates, with two iron-binding sites, resulted from a gene duplication.

A Mono-sited Transferrin From a Representative Deuterostome: The Ascidian Pyura stolonifera (Subphylum Urochordata)

An iron-binding protein has been found in the plasma of Pyura stolonifera. This protein has a molecular weight of about 41,000 +/- 2,000 and binds 1 mol iron/mol protein. The absorption maxima are lambda = 280 and lambda = 429 nm (E429/E280 = 0.044). Bicarbonate is bound concomitantly with high affinity and is necessary for optimal color formation at lambda = 429 nm. The protein showed a negligible exchange of iron with human apotransferrin under physiologic conditions over two hours. Upon incubation with rat reticulocytes, the protein reacts with membrane receptors for transferrins, and the protein, with its iron, is transported intracellularly where the iron is incorporated into heme. The 59Fe protein, after intravenous injection, disappears rapidly from the plasma and is excreted largely in the urine, with a substantial fraction present in the kidney and another large fraction present in the gut. These findings established the protein as a "transferrin" and support the concept that the larger transferrin molecule in vertebrates, with two iron-binding sites, resulted from a gene duplication.

Heterogeneity of the plasma iron pool: explanation of the Fletcher-Huehns phenomenon.

Observations on iron uptake by reticulocytes led Fletcher and Huehns to suggest differences in the behavior of the two iron-binding sites of the transferrin molecule. To clarify the continued controversy relating to this hypothesis, the original studies employing control plasma labeled with radioiron and the same plasma preincubated with reticulocytes were reexamined. Regardless of whether human or rabbit plasma was employed, there was a considerably higher uptake from the control plasma. Isoelectric focusing procedures and electrophoresis in urea gels revealed that the conspicuous difference in the amount of iron uptake is the result of differences in the proportion of di- to monoferric transferrin in the two plasmas and the competitive advantage of the diferric moiety. These studies provide an explanation for the Fletcher-Huehns phenomenon without invoking functional differences between the two sites for iron on the transferrin molecule.

Uptake and release of transferrin and iron by mitogen-stimulated human lymphocytes.

Phytohaemagglutinin stimulation of human peripheral blood lymphocytes resulted in the expression of transferrin receptors and the uptake of iron into the cells. As assessed from the resistance of the 125I label to pronase, transferrin was rapidly bound and internalized at 37 degrees C, while at 4 degrees C 85% of the 125I label remained on the cell surface and was degraded by the pronase. Over 94% of the 125I label associated with and subsequently released from the cells was acid precipitable, indicating that transferrin was not degraded during its uptake and release. After reincubation for 1 h in fresh medium 70% of the cell associated 125I-transferrin was released. In contrast, less than 30% of the 59Fe was released, showing that iron was removed from transferrin and retained by the cells. Concentration dependent binding of 125I-transferrin estimated at 37 degrees C occurred with an apparent Ka of 5.7 +/- 1.1 X 10(7) l mol-1 (mean +/- SD, n = 4) indicating little variation between cells from different individuals, although the number of transferrin molecules associated with the cells varied greatly from 6.2 X 10(4)/cell to 1.4 X 10(5)/cell. The rate of iron uptake from 59Fe and 125I labelled transferrin at 37 degrees C by the cells from different subjects was also very variable, with a range between 0.46 and 2.27 pg Fe/min/10(6) cells (n = 6). However, iron uptake did not correlate with the amount of transferrin bound. This suggests that transferrin uptake and the release of iron from the transferrin to the interior of the cell are controlled independently.

Rapid resolution of transferrin C subtypes through isoelectric focusing with 2-mercaptoethanol

The technique of choice currently used for the detection of serum transferrin molecular polymorphism is isoelectric focusing on polyacrylamide slab gels. However, this procedure is unsatisfactory for routine purposes, since a long pretreatment of the serum with iron-donor compounds or neuraminidase is necessary in order to obtain a complete resolution of the transferrin molecule. A very fast and highly economical standardized procedure for transferrin typing which enables a fair molecular resolution within only 3 1 2 h is reported. Protracted pretreatment of serum with neuraminidase or with iron-donor compouds can be totally avoided. An ultrathin layer of polyacrylamide gel is employed for the run, using pH ranges of 4–6.5 or 5–7. A short pretreatment of serum with a 13% solution of 2-mercaptoethanol is performed before the samples are placed on the gel. This technique has been used to perform transferrin typing in 396 cord serum samples from newborn infants of Arezzo (Tuscany), without occurrence of artifacts or the appearance of extra bands in transferrin patterns.

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes.

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.

DISTRIBUTION OF TRANSFERRIN AND TRANSFERRIN RECEPTORS IN THE RABBIT PLACENTA

The quantity and distribution of transferrin and transferrin-binding sites in the placenta were investigated in rabbits on the 28th-29th days of pregnancy. The animals were injected intravenously with a mixture of 59Fe-125I-labelled rabbit diferric transferrin and 131I-labelled rabbit albumin. The binding of transferrin to placentas removed 3-75 min later was determined by using the 131I-labelled albumin values to correct for tissue content of plasma. Mean values for transferrin binding of 1460 and 560 micrograms/g tissue were obtained 3-15 and 45-75 min after injection, respectively. Gel filtration of placental extracts prepared with the non-ionic detergent, Teric 12A9, showed that the 125I-labelled transferrin bound to a large molecular weight component which had the properties of a specific receptor. The receptor had a higher affinity for diferric transferrin than for apotransferrin. The subcellular distribution of transferrin binding sites was determined by differential centrifugation of placental homogenates and by electron microscope autoradiography. The results with the former method indicated that the transferrin was bound to the microsomal fraction of the cells. Autoradiography showed that the majority of the transferrin molecules were at intracellular sites, mainly on the membrane of intracellular vesicles. It is concluded that iron-containing transferrin molecules enter the trophoblast cells by endocytosis or via a canalicular system after binding to cell membrane receptors. The higher affinity of the receptors for diferric transferrin than for apotransferrin explains the difference in amount of transferrin binding found within 15 min of injecting labelled diferric transferrin and that found 45-75 min later when much of the iron had been removed from the transferrin.

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.

The primary structure of human serum transferrin. The structures of seven cyanogen bromide fragments and the assembly of the complete structure.

The amino acid sequences of seven cyanogen bromide fragments of human serum transferrin have been determined, and the primary structure of transferrin established by determining the order of these and three additional fragments (Sutton, M. R., MacGillivray, R. T. A., and Brew, K. (1975) Eur. J. Biochem. 51, 43-48) in the polypeptide chain. The order of the fragments was deduced from peptides that overlap methionyl residues which were obtained by thermolysin digestion of performic acid-oxidized transferrin or by partial peptic hydrolysis of unmodified transferrin, together with other evidence. The polypeptide chain of transferrin contains 679 amino acid residues, which together with the two N-linked oligosaccharide chains gives a calculated molecular weight of 79,570. Transferrin consists of two homologous domains (residues 1-336, 337-679), each associated with a single Fe-binding site, with both sites of glycosylation in the carboxyl-terminal domain at positions 413 and 611. Consideration of the primary structure in relation to previously published results provides information concerning the evolutionary development of transferrins and related proteins, and the locations of metal-binding residues in the transferrin molecule.

Structure and function of the transferrin receptor--a possible role in the recognition of natural killer cells.

The monoclonal antibody OKT9 reacts specifically with the receptors for transferrin in human cells and has been used to isolate and characterise this receptor [1]. The receptor is a dimeric glycoprotein (Mr = 180,000) composed of two apparently identical subunits (Mr = 90,000) which are disulphide linked. The transferrin receptor appears to be a transmembrane molecule and is phosphorylated, the phosphate group being predominantly on serine residues. The cell surface form of the molecule possesses both complex and high mannose oligosaccharide chains, which do not appear to have a direct role in antibody (OKT9) binding. The molecule can be cleaved from the cell surface into a 70,000 molecular weight fragment, suggesting that the major part of the receptor is exposed to the extracellular environment. The released 70,000 molecular weight fragments are not disulphide linked and possess antibody (OKT9) binding sites. Cross-linking studies using radiolabelled transferrin suggest that two molecules of transferrin are bound to each 180,000 molecular weight receptor dimer. In addition, each 70,000 molecular weight fragment can independently bind one molecule of transferrin (Fig. 1).

Iron transport and accumulation in mammalian cells

Iron is essential for all cells and mammalian cells are no exception. The daily requirements for iron are provided to a small extent from absorption of dietary iron essentially in their upper part of the intestine, to a large extent mediated by mucosal transferrin, and by reutilisation of tissue iron, to a major degree furnished by the breakdown of effete red blood cells, and assured by the serum β-globulin transferrin. In fact of the 35 mg or so or iron which exchanges between different body compartments each day, only 1 mg is contributed by mucosal iron absorption, and this latter is compensated by an equivalent amount of iron excretion (also about 1 mg/day). We shall concern ourselves here with three aspects of iron metabolism in mammalian cells: (i) iron uptake by cells from serum tranferrin (ii) accumulation of iron in the intracellular storage protein ferritin (iii) intracellular transport of iron to sites where it is required for essential cellular functions such as haem synthesis. Iron uptake from serum transferrin. It has been well established for many years that many mammalian cells (indeed probably all cells) have plasma membrane receptors for transferrin, and the transferrin receptor has recently have been purified and characterised [1]. It is an integral membrane protein of MW 180.000 composed of two disulphide linked subunits which are each capable of binding one transferrin molecule (MW 80.000 bearing one or two iron atoms). Subsequent to binding to its receptor, the transferrin-receptor complex is internalised by endocytosis and its iron is released by protonation of the bicarbonate (or carbonate) anion bound to the iron atom [2]. The apotransferrin molecule is recycled to the plasma membrane and released for reutilisation. In some cell types an additional mechanism intervenes in which the transferrin molecule is digested in lysosomes subsequent to iron release. Accumulation of iron in ferritin. The intracellular iron storage protein is ferritin in which provides a bio-available, soluble and non-toxic form of iron essentially as hydrolysed ferric oxyhydroxide enclosed within a globular protein shell (apoferritin) of MW 480.000. The amino acid sequences of human and horse apoferritins have been determined [3, 4] and the X-ray structure of horse spleen protein is well advanced [5]. The mechanism of iron deposition involves catalysis by the protein of the oxidation of Fe II bound to adjacent subunits, which may involve peroxo-bridged and μ-oxo intermediates prior to hydrolysis. Subsequent events may occur at the polymer surface or be catalysed by the protein and our current understanding of the incorporation of iron in ferritin will be reviewed. Intracellular Iron Transport for Haem Synthesis. The ferrochelatase of the mitochondrial matrix catalyses the final step in haem biosynthesis, namely the incorporation of Fe II into protoporphyrin IX. Recent studies suggest [6,7] that ferritin may well be the source of ferrous iron for haem synthesis via reduction of ferritin iron by reduced flavins generated by electrons from the respiratory chain, and implying a rather specific interaction of ferritin with the mitochondria. Recent result in this area will be presented and the role of ferritin in intracellular iron transport will be reviewed.

Partial purification of myoblast proliferation promoting factor from chick embryo extract

Chick embryo extract (EE) has been widely employed as a growth-promoting supplement in avian myogenic cell cultures. We have shown that transferrin and hypoxanthine-related molecules are essential components of EE for cell growth.

Transferrin receptors of human fibroblasts. Analysis of receptor properties and regulation.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.