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.     

The effect of serum and experimental variables on the transferrin and reticulocyte interaction

The transfer of iron from transferrin to the developing erythrocyte is a research area of high interest and considerable controversy. We have found that the results of transferrin-reticulocyte incubation studies are quite sensitive to the experiemental procedures that are utilized. Reticulocytosis has been induced in rabbits by phelbotomy and phenylhydrazine inhections. While the latter gives a higher reticulocyte count, the cells appear to exhibit an altered transferrin-membrane interaction. Transferrin has been iodinated by published methods utilizing chloramine-T and molecular iodine. The iodotransferrin products exhibit the same iron donation ability, however, evidence was found that the chloramine-T treatment leads to a nonspecific binding of transferrin to the reticulocyte. The means of saturating transferrin with ⁵⁹Fe is also of prime importance. Fe(NH₄)₂ (SO₄)₂ and especially FeCl₃ were found to yield non-specifically bound iron when added to transferrin or serum. This artifact was reflected in an altered transferrin-reticulocyte interaction. Using what we believe to be optimal conditions, the effect of serum on the transferrin-reticulocyte system was re-examined. The results clearly indicated an enhancement of iron uptake by reticulocytes in the presence of serum, as well as an accelerated incorporation of iron by the cytoplasmic fraction.     

Role of plasma membrane phospholipids in the uptake and release of transferrin and its iron by reticulocytes

Summary The involvement of membrane phospholipids in the utilization of transferrinbound iron by reticulocytes was investigated using [⁵⁹Fe]- and [¹²⁵I]-labelled transferrin and rabbit reticulocytes which had been incubated with phospholipas A. Transferrin and iron uptake and release were all inhibited by phospholipas A which produced a marked decrease in the relative abundance of phosphatidylcholine and phosphatidylethanolamine and equivalent increases in their lyso-compounds in the reticulocyte plasma membrane. There was a close correlation between the iron uptake rate and the rate and amount of transferrin uptake and the amount of the lysophospholipids in the membrane. Incubation of the cells with exogenous lysophosphatidylethanolamine or lysophosphatidylcholine also produced inhibition of iron and transferrin uptake. The reduced uptake produced by phospholipase A could be reversed if the lyso-compounds were removed by fatty acid-free bovine serum albumin or by reincubation in medium 199. Treatment with phospholipase A was shown to increase the amount of transferrin bound by specific receptors on the reticulocyte membrane but to inhibit the entry of transferrin into the cells.The present investigation provides evidence that the phospholipid composition of the cell membrane influences the interaction of transferrin with its receptors, the processes of endocytosis and exocytosis whereby transferrin enters and leaves the cells, and the mechanism by which iron is mobilized between its binding to transferrin and incorporation into heme. In addition, the results indicate that phosphatidylethanolamine is present in the outer half of the lipid bilayer of reticulocyte membrane.     

Factors affecting iron and transferrin release from rabbit reticulocyte ghosts to cytosol

⁵⁹Fe- and ¹²⁵I-labelled transferrin-labelled rabbit reticulocyte ghosts were incubated at 37°C for 60 min with unlabelled reticulocyte and erythrocyte stroma-free haemolysates, and the ability of these haemolysates to release ⁵⁹Fe- and ¹²⁵I-labelled transferrin was investigated. Reticulocyte and erythrocyte haemolysates were equally effective in releasing ⁵⁹Fe from the ghosts, but only the reticulocyte haemolysate was able to release ¹²⁵I-labelled transferrin. The elution profiles of the post-incubation haemolysates upon AcA 44 gel filtration were similar. The ⁵⁹Fe appeared as five separate peaks and the ¹²⁵I-labelled transferrin appeared as a single, unbound peak. In the post-incubation reticulocyte haemolysate, 25% of the ⁵⁹Fe was bound to ferritin and transferrin, and 69% was associated with the haemoglobin fraction; 52.8% of the ⁵⁹Fe was present as haem-⁵⁹Fe intimately associated with haemoglobin. Another 12.5% of the ⁵⁹Fe was loosely bound to proteins in the haemoglobin fraction. The haem-⁵⁹Fe released to the haemoglobin fraction was derived from preformed haem in the reticulocyte ghost. ⁵⁹Fe release was not impaired in experiments in which haem and protein synthesis were inhibited with isonicotinic acid hydrazide and cycloheximide. When tested alone, the haemoglobin fraction was able to release ⁵⁹Fe from the ghosts to an even greater degree than reticulocyte haemolysate. It is concluded that protein in the haemoglobin fraction function as heme carriers.Less than 6% of the ⁵⁹Fe released by reticulocyte haemolysate was associated with a low molecular size protein fraction. Removal of this fraction from the unlabelled haemolysate by ultrafiltration did not impair the ⁵⁹Fe-releasing capacity of the haemolysate. However, both this fraction and the ferritin fraction were able to bind some ⁵⁹Fe from the ghosts. Ferrous and ferric chelators, as well as defatted bovine serum albumin, were also able to bind ⁵⁹Fe from the ghosts, but not to the same degree as the haemolysates.The release of ¹²⁵I-labelled transferrin from the ghosts by the reticulocyte haemolysate was affected by stimulatory and inhibitory factors. The stimulatory factor(s) was present in the non-haemoglobin components of the haemoglobin fraction. The inhibitory effect was dependent on the low molecular weight fraction.     

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.     

Gallium uptake by transferrin and interaction with receptor 1

The kinetics and thermodynamics of Ga(III) exchange between gallium mononitrilotriacetate and human serum transferrin as well as those of the interaction between gallium-loaded transferrin and the transferrin receptor 1 were investigated in neutral media. Gallium is exchanged between the chelate and the C-site of human serum apotransferrin in interaction with bicarbonate in about 50 s to yield an intermediate complex with an equilibrium constant K ₁ = (3.9 ± 1.2) × 10⁻², a direct second-order rate constant k ₁ = 425 ± 50 M⁻¹ s⁻¹ and a reverse second-order rate constant k ₋₁ = (1.1 ± 3) × 10⁴ M⁻¹ s⁻¹. The intermediate complex loses a single proton with proton dissociation constant K _1a = 80 ± 40 nM to yield a first kinetic product. This product then undergoes a modification in its conformation which lasts about 500 s to produce a second kinetic intermediate, which in turn undergoes a final extremely slow (several hours) modification in its conformation to yield the gallium-saturated transferrin in its final state. The mechanism of gallium uptake differs from that of iron and does not involve the same transitions in conformation reported during iron uptake. The interaction of gallium-loaded transferrin with the transferrin receptor occurs in a single very fast kinetic step with a dissociation constant K _d = 1.10 ± 0.12 μM and a second-order rate constant k _d = (1.15 ± 0.3) × 10¹⁰ M⁻¹ s⁻¹. This mechanism is different from that observed with the ferric holotransferrin and suggests that the interaction between the receptor and gallium-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of gallium incorporation by the transferrin receptor-mediated iron-acquisition pathway is discussed.     

The effect of serum and experimental variables on the transferrin and reticulocyte interaction.

The transfer of iron from transferrin to the developing erythrocyte is a research area of high interest and considerable controversy. We have found that the results of transferrin-reticulocyte incubation studies are quite sensitive to the experimental procedures that are utilized. Reticulocytosis has been induced in rabbits by phelbotomy and phenylhydrazine injections. While the latter gives a higher reticulocyte count, the cells appear to exhibit an altered transferrin-membrane interaction. Transferrin has been iodinated by published methods utilizing chloramine-T and molecular iodine. The iodotransferrin products exhibit the same iron donation ability, however, evidence was found that the chloramine-T treatment leads to a nonspecific binding of transferrin to the reticulocyte. The means of saturating transferrin with 59Fe is also of prime importance. Fe(NH4)2(SO4)2 and especially FeCl3 were found to yield nonspecifically bound iron when added to transferrin or serum. This artifact was reflected in an altered transferrin-reticulocyte interaction. Using what we believe to be optimal conditions, the effect of serum on the transferrin-reticulocyte system was re-examined. The results clearly indicated an enhancement of iron uptake by reticulocytes in the presence of serum, as well as an accelerated incorporation of iron by the cytoplasmic fraction.     

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     

Relationship between pinocytic rate and uptake of transferrin by suspended rat hepatocytes

Summary The aim of this study was to compare quantitatively the capacity to transcytose (i.e. to uptake and release) transferrin (Tf) with the pinocytic activity of suspended adult rat hepatocytes. An oligodisperse preparation of¹³¹I-polyvinylpyrrolidone (PVP;M r 36000) was used to measure the inward and outward aspects of the pinocytic process in separate experiments. Cell association of rat¹²⁵I-Tf was measured at Tf concentrations approaching physiological, where⁵⁹Fe uptake obeyed first-order kinetics. Release studies with both PVP and Tf were carried out under conditions which minimized the probability ofde novo endocytosis of a molecule already released. Sets of experimental points representing cell-associated radioactivities were converted into continuous algebraic functions by fitting with two-term (release studies) or three-term (uptake studies) exponential equations. Transport of PVP and Tf through the cells was computed from these equations by deconvolution. This analysis showed that, under the present experimental conditions, the fractional transcytosis rates of Tf and PVP by hepatocytes were in the ratio of I:0.77. These values imply that, in the physiological range of Tf concentrations, about 75% of the Fe taken up by hepatocytes may be due to a pinocytic mechanism (fluid-phase or mixed). Inclusion of chloroquine (1 mM) in the suspending medium, both in uptake and release experiments, resulted in more PVP and Tf passing through the cells, while Fe uptake was reduced. It is suggested that the base probably exerted its enhancing effect on transcytosis by shunting the subcellular transport of PVP and Tf to the outward leg through a shorter circuit.Abbreviations BSA bovine serum albumin - HBSS Hank's balanced salt solution - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MEM minimal essential medium - PVP polyvinylpyrrolidone - Tf transferrin     

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.     

Acquisition of iron from transferrin regulates reticulocyte heme synthesis

Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59Fe from [59Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [59Fe]transferrin. Also, Fe-SIH stimulates [2-14C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59Fe incorporation into heme from either [59Fe]transferrin or [59Fe]SIH but does reverse the inhibition of 59Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes.     

Factors affecting cation-anion uptake balance and iron acquisition in peanut plants grown on calcareous soils

Dicotyledonous plants subjected to Fe-deficiency stress can decrease pH in the rhizosphere by proton excretion and reduce ferric iron by an activated reduction system in the plasma membranes of the root or by reductants released from the roots. The efficiency by which these plants take up Fe may strongly depend on their cation-anion balance. This study presents results of two experiments conducted to evaluate the effect of K, growth stage and cultivar on ionic balance and Fe acquisition of peanut (Arachis hypogaea L.) plants.Potassium applications to the high calcareous soil (30.3% CaCO₃) favoured proton release, but did not ameliorate plant Fe acquisition. At the earliest stages of plant growth, anion uptake exceeded cation uptake due to intensive N uptake. With time, a shift in the ionic balance was observed as a result of predominant cation uptake. It appears that the relationship between H/OH-ion release and Fe nutrition of peanut plants is actually a complex phenomenon under soil conditions and depends on some soil parameters, such as CaCO₃ content. Even by enhanced H-ion release Fe nutrition of plants can be impaired if soil CaCO₃ is too high.     

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

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

Ferric pyridoxal isonicotinol hydrazone can provide iron for heme synthesis in reticulocytes

We have examined whether reticulocytes depleted of transferrin might incorporate ⁵⁹Fe from ⁵⁹Fe-labelled pyridoxan isonicotinoyl hydrazone (PIH). Transferrin-depleted reticulocytes showed a time-, temperature- and concentration-dependent incorporation of ⁵⁹Fe when incubated with 20–200 μM ⁵⁹Fe-PIH. The amount of ⁵⁹Fe incorporated with 200 μM ⁵⁹Fe-PIH is equal to or higher than that taken up from transferrin at 20 μM ⁵⁹Fe concentration. After 60 min about 60% of the ⁵⁹Fe taken up by the cells is recovered in heme while the remainder is probably still bound to PIH. 1 mM succinyl acetone (a specific inhibitor of heme synthesis) inhibits PIH-mediated incorporation of ⁵⁹Fe into heme by about 79% indicating that ⁵⁹Fe from ⁵⁹Fe-PIH is incorporated into de novo synthesized protoporphyrin. As is the case with transferrin, erythrocytes do not incorporate ⁵⁹Fe from ⁵⁹Fe-PIH. Pretreatment of reticulocytes with pronase does not inhibit their ability to incorporate ⁵⁹Fe from ⁵⁹Fe-PIH, suggesting that, unlike the uptake of Fe from transferrin, membrane receptors are not involved in the uptake of Fe-PIH by the cells.     

Kinetics of iron passage through subcellular compartments of rabbit reticulocytes

Summary The kinetics of the separate processes of Fe₂(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and⁵⁹Fe,¹²⁵I-labeled rabbit transferrin. The binding of⁵⁹Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2×10⁵ m ^–1 sec^–1. The rate of internalization of⁵⁹Fe-transferrin was directly measured at 520±100 molecules of Fe₂(III)-transferrin internalized/sec/cell with 250±43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe₂(III)-transferrin internalization the flux of⁵⁹Fe was followed through three compartments: internalized transferrin, membrane, and cytosol.A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17±2 sec and 34±5 sec, respectively. Apparent rate constants of 0.0075±0.002 sec^–1 and 0.0343±0.0118 sec^–1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112±0.0025 sec^–1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe₂(III)-transferrin that are distinct from the handling of transferrin.     

Mechanism of Fe uptake by the leaf symplast: Is Fe inactivation in leaf a cause of Fe deficiency chlorosis?

The mechanism of iron (Fe) uptake from the leaf apoplast into leaf mesophyll cells was studied to evaluate the putative Fe inactivation as a possible cause of Fe deficiency chlorosis. For this purpose, sunflower (Helianthus annuus L.) and faba bean plants (Vicia faba L.) were precultured with varied Fe and bicarbonate (HCO ₃ ^- ) supply in nutrient solution. After 2–3 weeks preculture, Fe^III reduction and ⁵⁹Fe uptake by leaf discs were measured in solutions with Fe supplied as citrate or synthetic chelates in darkness. The data clearly indicate that Fe^III reduction is a prerequisite for Fe uptake into leaf cells and that the Fe nutritional status of plants does not affect either process. In addition, varied supply of Fe and HCO ₃ ^- to the root medium during preculture had no effect on pH of the xylem sap and leaf apoplastic fluid. A varied pH of the incubation solution had no significant effect on Fe^III reduction and Fe uptake by leaf discs in the physiologically relevant pH range of 5.0–6.0 as measured in the apoplastic leaf fluid. It is concluded that Fe inactivation in the leaf apoplast is not a primary cause of Fe deficiency chlorosis induced by bicarbonate. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.     

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

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