Effect of pH and iron content of transferrin on its binding to reticulocyte receptors

The effect of pH on the binding of apotransferrin and diferric transferrin to reticulocyte membrane receptors was investigated using rabbit transferrin and rabbit reticulocyte ghosts, intact cells and a detergent-solubilized extract of reticulocyte membranes. The studies were performed within the pH range 4.5–8.0. The binding of apotransferrin to ghosts and membrane extracts and its uptake by intact reticulocytes was high at pH levels below 6.5 but decreased to very low values as the pH was raised above 6.5. By contrast, diferric transferrin showed a high level of binding and uptake between pH 7.0 and 8.0 in addition to binding only slightly less than did apotransferrin at pH values below 6.5. It is proposed that the high affinity of apotransferrin for its receptor at lower pH values and low affinity at pH 7.0 or above allow transferrin to remain bound to the receptor when it is within acidic intracellular vesicles, even after loss of its iron, but also allow ready release from the cell membrane when it is exteriorized by exocytosis after iron uptake. The binding of transferrin to the receptor throughout the endocytosis-exocytosis cycle may protect it from proteolytic breakdown and aid in its recycling to the outer cell membrane     

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.     

Contrasting Effects of Polycations on Plaquing Efficiency of Encephalomyocarditis Virus Variants

Polycation treatment of L cell monolayers affected plaquing efficiency of both the r(+) and r variants of the encephalomyocarditis virus. Plaque formation by r(+) variant was decreased markedly by three structurally different types of synthetic basic polymers, diethylaminoethyl dextran, hexadimethrene (polybrene), and basic polyamino acids. In contrast, these same substances increased substantially the number of plaques formed by the r variant. The effect on the two variants was observed when polycations were applied to the cells before or simultaneously with the introduction of virus. The molar concentration and size of the polymer proved important. Thus, basic polyamino acids of low molecular weight were significantly more inhibitory for the r(+) variant than were those of high molecular weight. On the other hand, plaquing efficiency of the r variant was increased by relatively large polyamino acids, but not by polymers of small size. Basic polyamino acids inhibited r(+) plaque formation to a greater degree at low than at high pH values. However, plaquing efficiency of the r variant in polycation-treated cultures was not affected by changes in pH. Basic polymers appear to bind to cell membranes and affect either attachment or uptake of the viruses. The evidence suggests that the substances influence by different mechanisms the interaction of the r(+) and r variants with cells.     

Differences between human and rabbit transferrins

Rabbit reticulocyte incorporation of iron from rabbit transferrin was independent of transferrin iron saturation but uptake from human transferrin was saturation dependent. Unlike human transferrin, rabbit transferrin does not surrender its iron from any unique preferred iron-binding site and can be described as functionally homogeneic.The two proteins also differ in their acid-base iron-binding properties. One human transferrin iron binding site retains an ability to bind iron at somewhat acid pH but this property is not shared by rabbit transferrin.     

Differences between human and rabbit transferrins.

Rabbit reticulocyte incorporation of iron from rabbit transferrin was independent of transferrin iron saturation but uptake from human transferrin was saturation dependent. Unlike human transferrin, rabbit transferrin does not surrender its iron from any unique preferred iron-binding site and can be described as functionally homogeneic. The two proteins also differ in their acid-base iron-binding properties. One human transferrin iron binding site retains an ability to bind iron at somewhat acid pH but this property is not shared by rabbit transferrin.     

The effects of an antibody to the rat transferrin receptor and of rat serum albumin on the uptake of diferric transferrin by rat hepatocytes

The role of high-affinity specific transferrin receptors and low-affinity, non-saturable processes in the uptake of transferrin and iron by hepatocytes was investigated using fetal and adult rat hepatocytes in primary monolayer culture, rat transferrin, rat serum albumin and a rabbit anti-rat transferrin receptor antibody. The intracellular uptake of transferrin and iron occurred by saturable and non-saturable mechanisms. Treatment of the cells with the antibody almost completely eliminated the saturable uptake of iron but had little effect on the non-saturable process. Addition of albumin to the incubation medium reduced the endocytosis of transferrin by the cells but had no significant effect on the intracellular accumulation of iron. The maximum effect of rat serum albumin was observed at concentrations of 3 mg/ml and above. At a low incubation concentration of transferrin (0.5 microM), the presence of both rat albumin and the antibody decreased the rate of iron uptake by the cells to about 15% of the value found in their absence, but to only 40% when the diferric transferrin concentration was 5 microM. These results confirm that the uptake of transferrin-bound iron by both fetal and adult rat hepatocytes in culture occurs by a specific, receptor-mediated process and a low-affinity, non-saturable process. The low-affinity process increases in relative importance as the iron-transferrin concentration is raised.     

Binding of transferrin and metal ions by suspensions of reticulocyte-rich rabbit blood

Reticulocyte binding of Fe(III)_-transferrin and transferrin complexes with other metal ions have been compared by different investigators. The functional relevance of this comparison is not clear, therefore transferrin complexes with Fe(III), Cu(II), Mn(II) and Zn(II) have been studied further by DEAE-cellulose chromatography and by measurement of transferrin and metal uptakes by rabbit reticulocytes.Human Fe-transferrin behaved as a weaker anion than apotransferrin during DEAE-cellulose chromatography; since Fe-transferrin has a higher negative charge than apotransferrin and behaves a as stronger anion in electrophoretic systems, the chromatographic result was the opposite of that anticipated. The lower affinity of human Fe-transferrin for DEAE-cellulose is probably caused by a redistribution of charged groups on the surface of transferrin molecules when Fe(III) ions are bound and is therefore considered to be dependent on molecular conformation. Apotransferrin and divalent metal-transferrin complexes were found to have nearly equal affinities for DEAE-cellulose, thus the effect on surface charge of human transferrin molecules induced by binding Fe(III) appeared to be limited to that metal ion.Iron uptake by reticulocytes was associated with increased binding of transferrin to the cell surface: uptake of divalent metals occured without a concomitant increase in transferrin uptake or evidence of a specific metal-transfer process. Cu-transferrin was rapidly dissociated during incubation with cells.The effect of Fe(III)_binding on human transferrin molecules was to alter the molecular affinity for charged surfaces, namely DEAE-cellulose and reticulocyte membranes. This was less apparent with rabbit transferrin. Transferrin complexes with divalent metals behaved as apotransferrin in the process of association with reticulocytes.     

The rate limiting step in the reticulocyte uptake of transferin and transferrin iron Effects of some incubation variables

Reticulocytes incubated in an isotonic NaCl saline medium containing glucose, glutamine and amino acids, were able to detach both iron atoms from all the transferrin incorporated by them. In the absence of these metabolites, although transferrin uptake was the same, the reticuloctes failed to remove completely the iron from the transferrin which they incorporated.It has been shown before that there is unspecific as well as specific binding of transferrin to the reticulocyte. By incubating the cells in the presence of a high concentration of bovine serum albumin, we have been able to prevent the unspecific attachment of transferrin.At least 94% of the iodinated transferrin was capable of donating its iron to the reticulocytes.     

The rate limiting step in the reticulocyte uptake of transferrin and transferrin iron. Effects of some incubation variables.

Reticulocytes incubated in an isotonic NaCl saline medium containing glucose, glutamine and amino acids, were able to detach both iron atoms from all the transferrin incorporated by them. In the absence of these metabolites, although transferrin uptake was the same, the reticulocytes failed to remove completely the iron from the transferrin which they incorporated. It has been shown before that there is unspecific as well as specific binding of transferrin to the reticulocyte. By incubating the cells in the presence of a high concentration of bovine serum albumin, we have been able to prevent the unsepcific attachment of transferrin. At least 94% of the iodinated transferrin was capable of donating its iron to the reticulocytes.     

Reconstitution of the transferrin receptor in lipid vesicles. Effect of cholesterol on the binding of transferrin

Purified rabbit reticulocyte transferrin receptors were incorporated into phosphatidylcholine vesicles containing varying amounts of cholesterol. The binding of transferrin to the receptor in the reconstituted vesicles had three distinct characteristics: (1) The binding of transferrin exhibited the two components characteristic of transferrin binding to erythroid cells, a saturable, specific component and a nonsaturable, nonspecific component. (2) Transferrin binding exhibited positive cooperativity at low cholesterol/phospholipid (C/P) molar ratios. However, the cooperativity diminished and then disappeared as the C/P molar ratios were increased to the levels found in circulating red blood cells. (3) The amount of specific transferrin binding to the reconstituted vesicles also decreased as the C/P molar ratio was increased. These results indicate that in the reconstituted system the lipid environment plays a significant role in the expression of transferrin receptors.     

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 59Fe 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 has 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-donating properties of human transferrin and identifies that the near neutral iron-binding site initially surrenders its iron to these cells.     

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.     

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.     

Binding of bismuth to serum proteins: implication for targets of Bi(III) in blood plasma

Bismuth complexes have been widely used in clinical treatment as antiulcer drugs. However, different adverse effects have been observed and the diagnosis is generally confirmed by the detection of bismuth in blood or blood plasma. In this study, binding of bismuth to human serum albumin was studied by fluorescence spectroscopy with the binding constant logK(a) to be 11.2. Competitive binding of bismuth to human albumin and transferrin was carried out at pH 7.4 by FPLC and ICP-MS. It was found that over 70% of bismuth binds to transferrin even in the presence of a large excess of albumin (albumin/transferrin=13:1) at pH 7.4, 10 mM bicarbonate. The distribution of bismuth between the two proteins was almost unchanged when Cys(34) of albumin was blocked. However, all bismuth binds to albumin when iron-saturated transferrin was used. Almost all of the bismuth was distributed over the fractions containing transferrin (70%) and albumin (<30%) in serum. The percentage of bismuth associated with transferrin was further increased by 15% with elevated transferrin in serum. Binding of bismuth to transferrin is much stronger than human albumin. Transferrin is probably the major target of bismuth in blood plasma, and it may play a role in the pharmacology of bismuth.     

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.     

Albumin and transferrin synthesis during development in the rat

In this study, the incorporation of [(14)C]leucine into albumin and transferrin in early rat foetuses, vitelline plus amniotic membranes, chorioallantoic placenta and perinatal rat liver slices was measured and used to detect and compare the rates of synthesis of the two proteins. Albumin synthesis was detected in the body of foetuses from 13 days gestation onwards. Transferrin synthesis was detected only after day 15. Transferrin synthesis was demonstrable in the membranes but not in the chorioallantoic placenta of all the animals investigated, i.e. from 13 to 19 days gestation. Synthesis of albumin and transferrin by the liver of near-term and postnatal animals was shown to correlate with published data on the parenchymal cell number/unit wet wt. of liver. Near-term foetuses synthesized relatively more transferrin than albumin when compared with 10-day postnatal animals. The serum concentrations of the two plasma proteins were also determined. These increased before term whereas the rate of synthesis of albumin and transferrin declined. Postnatally, plasma albumin concentration increased but transferrin concentration decreased, yet the rates of synthesis of both proteins by the liver increased with age. This lack of correlation between the rates of synthesis of the two proteins and their respective plasma concentrations could be explained in part by their increased stability after birth. There was also evidence that the liver haemopoietic cells took up transferrin although they do not synthesize the protein. Thus the decrease in this population of cells during development could also contribute to the discrepancy between liver synthesis and serum concentrations of transferrin.     

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 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.     

Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.     

Albumin inhibition of transferrin low-affinity binding to K562 cells

Several reports have suggested that variations of albumin concentration in the incubation medium can modulate the magnitude of transferrin binding to the cells. We have investigated this problem further using K562 cells. In the absence of human serum albumin, transferrin binding demonstrated a non-saturable curve which, upon Scatchard analysis, showed two components with high and low affinities. In the presence of 0.5% human serum albumin, the low-affinity but not the high-affinity component was totally inhibited and, thus, the binding showed a saturation plateau at transferrin concentration of 6 micrograms/ml. Increasing concentrations of human serum albumin in the incubation medium led to progressive inhibition of transferrin binding, reaching a plateau at 0.2% human serum albumin. At this concentration transferrin binding was about 12 ng/10(6) cells, corresponding to the saturation plateau for high-affinity binding. Low-affinity transferrin binding in the absence of human serum albumin could readily be displaced by subsequent addition of albumin. Similar inhibition was obtained by another serum protein, ceruloplasmin, suggesting that this inhibition is not unique to albumin and may be a common property of all proteins. Incubation at 37 degrees C with 59Fe-labeled transferrin indicated that all iron uptake occurs through high-affinity binding. We conclude that the reported variations in magnitude of transferrin binding by the cell due to variations in albumin concentration are the result of inhibition of low-affinity binding of transferrin by albumin.