Interaction Of Transferrin Research Articles

Nonclinical safety evaluation of pabinafusp alfa, an anti-human transferrin receptor antibody and iduronate-2-sulfatase fusion protein, for the treatment of neuronopathic mucopolysaccharidosis type II

Pabinafusp alfa is a fusion protein comprising a humanized anti-human transferrin receptor (TfR) antibody and human iduronate-2-sulfatase. It was developed as a novel modality to target central nervous system-related symptoms observed in patients with mucopolysaccharidosis type II (MPS II, also known as Hunter syndrome). As the fusion protein contains an entire IgG1 molecule that binds TfR, there may be specific safety concerns, such as unexpected cellular toxicity due to its effector functions or its ability to inhibit iron metabolism, in addition to general safety concerns. Here, we present the comprehensive results of a nonclinical safety assessment of pabinafusp alfa. Pabinafusp alfa did not exhibit effector functions, as assessed by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity studies in TfR-expressing hematopoietic cells. Repeat-dose toxicity studies in cynomolgus monkeys showed that pabinafusp alfa did not induce any significant toxicological changes at doses up to 30 mg/kg/week upon intravenous administration for up to 26 weeks. Interaction of transferrin with TfR was not inhibited by pabinafusp alfa, suggesting that the effect of pabinafusp alfa on the physiological iron transport system is minimal, which was confirmed by toxicity studies in cynomolgus monkeys. These findings suggest that pabinafusp alfa is expected to be safe for long-term use in individuals with MPS II.

Brownian motion-based nanoparticle sizing-A powerful approach for in situ analysis of nanoparticle-protein interactions.

A key hurdle toward effective application of nanoparticles (NPs) in biomedicine is still the incomplete understanding of the biomolecular adsorption layer, the so-called protein corona, which inevitably forms around NPs when they are immersed in a biofluid. NP sizing techniques via the analysis of Brownian motions offer a powerful way to measure the thickness of the protein corona in situ. Here, the fundamentals of three techniques, dynamic light scattering, fluorescence correlation spectroscopy, and nanoparticle tracking analysis are briefly summarized. Then, experimental procedures for the determination of binding curves are presented in a tutorial fashion. Nanoparticle sizing experiments are illustrated with a selection of recent results on the interactions of transferrin with hydrophilic and hydrophobic polystyrene nanoparticles, and key insights gained from this work are discussed.

CAMKK2-CAMK4 signaling regulates transferrin trafficking, turnover, and iron\xa0homeostasis

BackgroundCirculatory iron is a hazardous biometal. Therefore, iron is transported in a redox-safe state by a serum glycoprotein - transferrin (TF). Different organs acquire iron from the systemic circulation through a tightly regulated mechanism at the blood-tissue interface which involves receptor-mediated internalization of TF. Thus, abnormal TF trafficking may lead to iron dyshomeostasis associated with several diseases including neurodegeneration. Iron -induced toxicity can cause neuronal damage to iron-sensitive brain regions. Recently, it was discovered that CAMKK2, a calcium (Ca2+)/calmodulin-activated kinase, controls receptor-mediated TF trafficking in mouse tissues, specifically in the brain. The biological function of CAMKK2 is mediated through multiple downstream effectors. Both CAMKK2 and one of its downstream kinase, CAMK4, exhibit overlapping expression in mouse brain. The role of CAMK4 in vesicular transport has been reported and loss of CAMKK2 or CAMK4 leads to cognitive defects in mouse. Therefore, it was hypothesized that CAMKK2-CAMK4 signaling regulates receptor-mediated TF trafficking and iron homeostasis which may be responsible for the neuronal malfunction observed in CAMKK2- or CAMK4-deficient mice.MethodsCAMK4−/− mouse was used to study tissue-specific turnover of TF, TF-receptor (TFRC) and iron. CRISPR/Cas9-based CAMKK2 and/or CAMK4 deleted human embryonic kidney-derived HEK293 cell clones were used to study the molecular defects in receptor-mediated TF trafficking. Further, a “zero functional G protein” condition in HEK293 cell was exploited to study CAMKK2-CAMK4 signaling-mediated regulation of intracellular Ca2+ homeostasis which was linked to calcium signaling during TF trafficking.ResultsLoss of CAMK4 leads to abnormal post-translational modifications (PTMs) and turnover of TF in mouse cerebellum and liver which was associated with iron dyshomeostasis in these tissues. The HEK293 cell-based study revealed that the absence of CAMKK2-CAMK4 signaling altered intracellular Ca2+ homeostasis and lead to abnormal calcium signaling during TF trafficking. Also, CAMKK2-CAMK4 signaling deficiency affected the molecular interaction of TF and TF-receptor-associated protein complexes which indicated a potential failure in the recruitment of interacting proteins due to differential PTMs in TF.ConclusionOverall, this study established a novel mechanistic link between intracellular Ca2+ level, receptor-mediated TF trafficking, and iron homeostasis, all regulated by CAMKK2-CAMK4 signaling.96jS1k-UhvBwj8b5MHadpoVideo Graphical abstract

Interaction of transferrin with non-cognate metals studied by native electrospray ionization mass spectrometry

Transferrin (Tf) is a key player in iron homeostasis which transports otherwise insoluble ferric ion and delivers it to cells via receptor-mediated endocytosis. Since Tf is also capable of binding a range of other metals with very high affinity, it remains in the focal point of multiple studies aiming at understanding the mechanism of biodistribution of clinically relevant non-ferric metals. The latter included toxic metals, as well as metals that can be exploited for medicinal or therapeutic benefit (e.g., radionuclides). More recently, there was a growing interest in using non-cognate metals as tracers of Tf-based therapeutics in animal-based disease models. In this work interaction of seven metal ions in their physiologically relevant oxidation states (Cr3+, In3+, Ti4+ and four lanthanides) with Tf were studied using native electrospray ionization mass spectrometry (ESI MS). Cr3+, In3+ and Ti4+ display Tf-binding properties that are similar to those of the cognate metal (Fe3+), while Tf association with lanthanides leads to formation of complexes that are very unstable in the gas phase, indicating the protein/metal binding mechanism and structure in this case are very different from those leading to formation of the Fe2Tf complex. Native ESI MS also allowed the receptor binding competence of Tf loaded with non-ferric metals to be evaluated. Lastly, behavior of these complexes under conditions mimicking the endosomal environment (where the release of the cognate metal actually occurs) was also evaluated. This analysis revealed striking similarities between Cr2Tf, In2Tf and Ti2Tf on one hand, and the diferric form of Tf on the other hand, strongly suggesting that Tf is indeed a critical player in homeostasis of these metals and must be considered both in the analysis of metal toxicity and in the design of metal-based therapeutics.

Interaction of transferrin with cefuroxime sodium

ABSTRACTThe interaction between cefuroxime sodium and transferrin in physiological buffer (pH 7.4) was investigated by spectroscopy at 298, 310, and 318 K. The analysis of data indicated that quenching mechanism of the transferrin–cefuroxime sodium system was probably static. The electrostatic force played an important role in the conjugation reaction between transferrin and cefuroxime sodium. The order of magnitude of binding constants (Ka) was 104, and the number of binding site (n) in the binary system was approximately equal to 1. Besides, the values of Hill’s coefficients were lager than 1, which indicated very weakly positive cooperativity in the transferrin–cefuroxime sodium system. The synchronous fluorescence spectra and circular dichroism spectra revealed that the microenvironment and the conformation of transferrin were changed during the binding reaction.

Lomefloxacin promotes the interaction between human serum albumin and transferrin: A mechanistic insight into the emergence of antibiotic's side effects

Human serum albumin (HSA) and serum transferrin (TF) are two drug carrier proteins in vivo. In this study it was investigated how lomefloxacin (LMF) binding affected the HSA–TF interaction using different spectroscopic, calorimetric and molecular modeling techniques. Fluorescence, circular dichroism and synchronous fluorescence revealed that LMF could bind to both proteins, resulting in protein conformational changes. Moreover, HSA and TF could interact so that some fluorescence residues were positioned at the interface and were shielded from quenching by LMF. The interaction between HSA and TF was further confirmed by fluorescence resonance energy transfer. Quantitative analyses of the far-UV CD spectra of the HSA–TF interaction in the presence and absence of LMF revealed secondary structural changes in detail. Resonance light-scattering studies demonstrated that the HSA–TF interaction resulted in a new species with a larger size, and that the presence of LMF could further favor this reaction. Isothermal titration calorimetry revealed that electrostatic interaction was dominant in the absence of LMF, whereas van der Waals forces and hydrogen bonding become significant in its presence. On the other hand, it was found that the binding constant of TF bound to HSA was stronger in the presence of LMF. ANS fluorescence further indicated that hydrophobic interactions play a minor part in the HSA–TF system. Molecular modeling studies confirmed the presence of fluorophore residues, hydrogen bonding and electrostatic interactions at the interface of the HSA–TF complex. It also suggested that the binding sites of LMF were not located there. These data indicate that LMF can modify the interaction between HSA and TF as two model proteins present in serum. The relevance to drugs’ side effects, pharmacokinetic of drugs and selection of diagnostic biomarker is discussed.

Corroles that bind with high affinity to both apo and holo transferrin

The interactions of transferrin (Tf) with the water soluble corrole 1 and with its gallium ( 1- Ga) and manganese ( 1- Mn) complexes were studied to establish the possible utilization of corrole-transferrin conjugates for targeting these corroles to cells that express the transferrin receptor. The protein, in both its iron-free apo form (apoTf) and the iron-bound holo form (holoTf), was found to spontaneously bind all three derivatives. This conclusion was reached from titrations followed by several spectroscopic methods and dilution experiments measured by fluorescence. The such elucidated very small dissociation constant of 2 × 10 −7 M and 3 × 10 −8 M for 1- Ga with apoTf and holoTf, respectively and <10 −9 M for 1 with both protein forms are clearly relevant for the physiological concentration of transferrin in serum.

Transferrin is required for early T-cell differentiation.

Transferrin, the major plasma iron carrier, mediates iron entry into cells through interaction with its receptor. Several in vitro studies have demonstrated that transferrin plays an essential role in lymphocyte division, a role attributed to its iron transport function. In the present study we used hypotransferrinaemic (Trf(hpx/hpx)) mice to investigate the possible involvement of transferrin in T lymphocyte differentiation in vivo. The absolute number of thymocytes was substantially reduced in Trf(hpx/hpx) mice, a result that could not be attributed to increased apoptosis. Moreover, the proportions of the four major thymic subpopulations were maintained and the percentage of dividing cells was not reduced. A leaky block in the differentiation of CD4(-) CD8(-) CD3(-) CD44(-) CD25(+) (TN3) into CD4(-) CD8(-) CD3(-) CD44(-) CD25(-) (TN4) cells was observed. In addition, a similar impairment of early thymocyte differentiation was observed in mice with reduced levels of transferrin receptor. The present study demonstrates, for the first time, that transferrin itself or a pathway triggered by the interaction of transferrin with its receptor is essential for normal early T-cell differentiation in vivo.

A brief history of brain iron research

Brain iron research began in the second half of the 19th century when Perls [1] described the histochemical visualization of ‘‘iron oxide’’ in tissues by exposure of sections to a mixture of potassium ferrocyanide (1%) and hydrochloric acid (1%). The reaction causes an alluring blue (‘‘Prussian Blue’’) and continues to be the main method for the staining of iron in tissues everywhere. It is simple and quite specific but lacks sensitivity, especially with paraffin-embedded tissues. Enhancement by subsequent incubation in a mixture of diaminobenzidine and hydrogen peroxide greatly improves sensitivity [2] and reveals reaction product in much greater detail, especially in optimally fixed brain [3]. The distinction of ‘‘heme-’’ and ‘‘non-heme-’’ iron in the brain must be attributed to Zaleski [4] who observed that the iron in hemoglobin did not react with potassium ferrocyanide, hence the blue color had to arise from non-heme iron. He also concluded that brain iron was present in linkage to a protein and that it was largely ferric rather than ferrous. This discovery antedated the recognition of ferritin as the main iron-carrying protein in brain by 69 years [5]. Guizzetti [6] and Spatz [7] used immersion of fresh or fixed animal and human brain slices in Perls’ solution to visualize iron in various gray and white matter structures of the brain. The long paper by Spatz [7] offered the first color illustrations of the unusual differential distribution of iron. The most intense macrostaining occurred in the globus pallidus, the red nucleus, and the substantia nigra (including the melanin-free pars reticularis), followed by the caudate nucleus, the putamen, the subthalamic nucleus, and the dentate nucleus of the cerebellum. The reaction was less intense in the thalamus but was still stronger than in the cerebral cortex and the subcortical white matter. In vibratome or frozen sections of normal rat and rabbit brain, the enhanced iron stain [2] shows iron in microglia, oligodendroglia, astrocytes (especially the tanycytes), nerve cells, and possibly even axons and myelin sheaths. The staining of oligodendroglia in the cerebral white matter is not uniform. Reaction product occurs in clusters of ironreactive cells, but the significance of this distribution is not known. Iron is readily lost from sections unless the tissue is fixed promptly by aldehydes or ethanol. One of the reasons is the high solubility of ferritin in aqueous solvents. As in other organs, brain ferritin contains light (L-) and heavy (H-) subunits, and its biosynthesis is subject to translational control by the iron-regulatory proteins 1 and 2 (IRP) [8– 10]. Ferritin is readily detected by immunocytochemistry or immunofluorescence. The distribution is quite similar to that of iron when detected by histochemistry with Perls’ [1] ingredients. Reaction product for IRP-1 (the ‘‘ferritin repressor protein’’ in Ref. [8]) occurs mostly in astrocytes [11]. Bloch et al. [12] showed that brain contains transferrin and transferrin messenger ribonucleic acid (mRNA). Antisera to transferrin yield reaction product that does not coincide with that of iron or ferritin [13], and there is considerable variability among species [14]. However, transferrin is abundant in the oligodendroglial cytoplasm of many mammals and has become a reliable biochemical and immunocytochemical marker for development and pathology of these cells. In addition to transferrin, brain also contains transferrin receptor protein (TfR) [15]. Antisera to TfR reveal reaction product in the endothelium where the protein may serve in iron uptake. However, the mechanism of brain iron homeostasis and exchange is likely much more complex than the interaction of transferrin and TfR on the surface of endothelial cells. In recent years, systematic investigations of intestinal iron absorption and hemochromatosis have led to the characterization of several proteins that accomplish iron transport across membranes of cells and organelles. While there is no universal agreement on their names, ‘‘divalent metal transporter 1’’ (DMT1) (syn. divalent cation transporter 1 [DCT1]; natural resistance-associated macrophage protein 2 [NRAMP2]) and ferroportin (syn. metal transporter protein 1 [MTP1], ironregulated transporter 1 [IREG1]) are now widely recognized (brief review in Ref. [16]). Immunocytochemistry reveals a wide distribution of DMT1 in neurons, glial cells, and the

Extracellular defence against oxidative stress in the newborn

An efficient antioxidant defence against reactive oxygen species requires the synergistic interaction between intracellular and extracellular antioxidants. The extracellular system is composed of preventive and chain-breaking antioxidants. The most important preventive extracellular antioxidants are transferrin and caeruloplasmin which protect against iron induced damage. The main chain-breaking antioxidants are vitamin C and E, uric acid, bilirubin, and sulfhydryl groups, but unidentified antioxidants also contribute to the total antioxidant capacity of extracellular fluids. Investigation into the clinical significance of these antioxidants has just started and many questions remain unanswered. In particular, the interaction of transferrin, caeruloplasmin, vitamin C and iron may be important in the pathogenesis of disease such as respiratory distress syndrome and hypoxic/ischaemic encephalopathy in the newborn.

Interaction of transferrin with rat alveolar macrophages.

Binding of rat transferrin to isolated alveolar macrophages was investigated in the 0.125 nM to 2 microM range. Computer analysis of the data revealed two classes of binding sites, a small number (< 1000 exposed/cell) having high affinity (dissociation constant (Kd), 3.4 nM) and a large number (approximately 4 x 10(6)/cell) having low affinity (Kd 48 microM). Measurements with a monoclonal antibody to the rat transferrin (rTf) receptor yielded values in the same range as the high-affinity sites derived from studies of ligand binding. Binding to the low-affinity sites at pH 5.8 was nearly one order of magnitude stronger than that at pH 7.3. Bovine lactoferrin (12 microM), cationized bovine serum albumin (14 microM), L-arginine (50 mM), and L-lysine (50 mM) did not compete against rTf binding to the low-affinity sites. Removal of an average of 2.6 x 10(8) sialyl residues from each cell did not affect binding. Heparan sulphate proteoglycan purified from alveolar macrophages bound strongly to immobilized rTf, thus raising the possibility that the low-affinity interaction of transferrin with these cells may be mediated, at least in part, by this glycosaminoglycan.

Interaction of transferrin saturated with iron with lung surfactant in respiratory failure.

Proteins that decrease the surface activity of surfactant accumulate in epithelial lining fluid in respiratory failure. The aim of this study was to isolate a surfactant inhibitor from the airways of rabbits in acute respiratory failure induced by bronchoalveolar lavage (BAL). This inhibitor was identified as being transferrin (TF). Unlike serum TF, TF recovered in respiratory failure was saturated with iron (Fe(3+)-TF). Fe(3+)-TF decreased the surface activity of normal surfactant in vitro, whereas iron-free TF had no effect. In the presence of H2O2 and a reducing agent, Fe(+3)-TF inactivated the surfactant complex: the surface absorption rate was decreased, immunoreactive surfactant protein A was decreased, and malondialdehyde was formed. The acute effects of Fe(3+)-TF and iron-free TF applied to the airways were studied in animal models. In respiratory failure induced by BAL, Fe(3+)-TF deteriorated respiratory failure, whereas iron-free TF had no effect. In respiratory failure induced by hyperoxia for 48 h, administration of iron-free TF ameliorated the respiratory failure and improved the surface activity in BAL. We propose that Fe(3+)-TF accumulating in epithelial lining fluid during lung damage contributes to surfactant inhibition and promotes the formation of free radicals that inactivate the surfactant system.

Interaction of transferrin and its iron-binding fragments with heparin.

The interaction of heparin with transferrin (Tf; bovine and rat) and the isolated iron-binding lobes of bovine Tf were investigated. Affinity chromatography of rat Tf on heparin-agarose showed that interaction depended on both the iron content of Tf and the pH of the medium. Both the iron-free and iron-saturated forms of Tf were strongly bound by the column at pH 5.6, but only the iron-free form revealed significant affinity at pH 7.4. Desialylation of Tf moderately promoted interaction, treatment with cyclohexanedione moderately reduced interaction, and succinylation abolished it altogether. In the presence of heparin, iron release from the N-terminal lobe of native bovine Tf was accelerated and from the C-terminal lobe it was slightly reduced. The heparin effect remained qualitatively the same on each lobe after their separation by tryptic digestion and DEAE-cellulose chromatography. The affinity of native bovine Tf for heparin was very close to that of its isolated N-terminal lobe, thus suggesting that it is this portion of the molecule that binds to the glycosaminoglycan. It is concluded that the consequences for iron-binding strength of the two transferrin lobes are diagonally opposite when Tf is bound to heparin as opposed to its natural cell-surface receptor.

Iron-transferrin binding to isolated guinea pig enterocytes and the regional localisation of intestinal iron transfer during ontogeny

Neonatal animals are iron replete but in comparison with adults they display increased intestinal iron absorption. In order to examine possible mechanisms for this developmental adaptation we have measured the appearance of iron in peripheral blood following 30 min exposure of duodenal and ileal segments of adult and neonatal guinea pigs in vivo to 59Fe-ascorbate. Parallel experiments have determined the kinetics of 125I-labelled diferric transferring binding to villus enterocytes isolated from duodenum and ileum. In adult animals the rate of appearance of 59Fe in peripheral blood was 11-fold greater following duodenal, compared to ileal exposure to the radioligand. No such regional difference was detected in the neonate. Isolated cells showed saturable binding of [ 125I]transferrin which was maximal between 30 and 60 min. The kinetics of specific transferrin binding by adult duodenal and ileal enterocytes were similar and were also not significantly different to respective values in neonatal duodenal and ileal cells. Thus, it is likely that increased iron absorption in the neonate is due in part to enhanced ileal iron transfer. The interaction of transferrin with its receptor, however, is not involved in this developmental change in uptake.

Two pathways for iron uptake by guinea pig reticulocytes

We have demonstrated that the intracellular processing of transferrin to effect iron removal involves two pathways, one sensitive to rotenone and the other not. We have also found that the effect of the rotenone is dependent on the transferrin concentration: iron uptake was suppressed with concentrations of transferrin in the micromolar range, and was not suppressed at physiologic concentrations of transferrin. Rotenone does not disturb transferrin's interaction with its extracellular receptor, indicating that its action must be intracellular. The following model is suggested: that separate pathways are entered by transferrin in the cell. The first pathway is preferentially utilized when transferrin is in short supply. It begins with an intracellular site which has a high affinity (and low capacity) for either iron or transferrin. The second pathway begins with an intracellular site which has a high capacity (but low affinity) for either iron or transferrin and is utilized when transferrin is in physiologic concentration (and the low-capacity, high-affinity site is saturated); the pathway it initiates is dominant when transferrin is abundant. We speculate that the high-affinity low-capacity pathway may serve to direct intracellular iron to sites which would be critically injured by iron excess.

Transferrin in FRTL5 Cells: Regulation of Its Receptor by Mitogenic Agents and Its Role in Growth*

Transferrin, a serum iron-binding protein, delivers iron to the cell after binding to specific receptors on the cell surface and is an important component of culture medium for virtually all cell lines, including the FRTL5 line of rat thyroid follicular cells. Therefore, we undertook studies in FRTL5 cells to examine the regulation of the transferrin receptor, the effects of transferrin on growth and differentiated functions, and the interactions of transferrin with several mitogenic pathways. FRTL5 cells possess one class of saturable transferrin receptors (Ka, 0.7 x 10(9) M-1). Binding of 125I-labeled transferrin was highest in actively growing cells and declined progressively, reaching minimal values when confluence was achieved. Removal of transferrin from culture medium caused a rapid increase in transferrin binding. TSH, acting within 5 min, induced a modest increase in transferrin binding, due to a cycloheximide-resistant increase in binding sites. Binding of transferrin after a 24-h incubation was also increased by other mitogenic agents, (Bu)2cAMP, forskolin (FK), insulin, insulin-like growth factor-I (IGF-I), and the phorbol ester TPA. Transferrin alone stimulated growth only minimally, but enhanced the mitogenic effect of TSH, (Bu)2cAMP, and FK, all of which act through the cAMP pathway. In contrast, transferrin did not alter the cAMP-independent mitogenic effects of insulin and IGF-I. Transferrin did not affect TSH-induced cAMP generation. Desferoxamine, an iron chelator, inhibited the mitogenic effects of all of the agents tested. Desferoxamine had no significant effect on TSH-induced cAMP accumulation. We conclude that FRTL5 cells contain saturable receptors for transferrin whose abundance varies with the rate of cell replication. Transferrin down-regulates its own receptors, while stimulation of growth by various mitogens is accompanied by increased binding of transferrin. Transferrin enhances the mitogenic effect of the cAMP-dependent mitogens, TSH, (Bu)2cAMP, and FK, without modifying basal or stimulated cAMP generation. In contrast, transferrin fails to affect the mitogenic responses to IGF-I and insulin, which are cAMP independent. Iron is required for the mitogenic response to various mitogens, especially those that are cAMP dependent.

Interaction of transferrin with dihydrolipoate and dihydrolipoate/iron complexes

Interaction of transferrin with dihydrolipoate and dihydrolipoate/iron complexes

Interaction of transferrin with iron-loaded rat peritoneal macrophages.

Rat peritoneal macrophages are capable, in vitro, of processing and releasing iron derived from phagocytosed, immunosensitized red cells. From 20% to 60% of the red cell iron can be returned to the culture medium in 24 h, with resident macrophages more active than inflammatory, peptone-induced macrophages. When apotransferrin is present in the culture medium, from 39% to 72% of iron released from macrophages is bound to the protein, with most of the remainder in a ferritin-like form. No distinct preference of released iron for either site of transferrin could be observed. The absence of apotransferrin depresses iron release only slightly, with much of the iron then released in a form readily available to the protein in vitro. Pronase treatment of macrophages, which abolishes their ability to bind transferrin, depresses iron release no more than 10-15%. It appears, therefore, that binding of apotransferrin to macrophages may not be essential for iron excretion by the cells.

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 1.14·10 5molecules/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 4.4·10 4±1.9·10 4 for cells in suspension and 6.6·10 4±2.3·10 4 for adherent cultured cells. The apparent association constants were 1.23·10 7 1·mol −1 and 3.4·10 6 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 Fe 11 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.

Interaction of transferrin with solubilized receptors from reticulocytes.

A chromatographic assay method was developed to measure solubilized transferrin receptors from reticulocytes. Binding of transferrin to its receptor was found to occur in a reversible manner involving only one type of receptor, viz. one which is readily saturated with, and has a high affinity for, transferrin. The results were consistent with a bimolecular interaction mechanism between transferrin and its receptor. A number of reticulocyte populations were examined and they contained about 100 000 receptors per cell with a receptor density of about 800 per μm2 of cell surface area. The equilibrium constant of association (Ka) of transferrin for its receptor was approximately 107 1 · mol−1. The binding process was markedly pH‐dependent, with a pH optimum of 7.8. Binding was considerably reduced when the ionic strength or calcium ion levels were below physiological values. Analysis of the binding process at different temperatures showed that ΔHo for the interaction was −444 J · mol−1, ΔG° was −44 · 3 kJ · mol−1 and ΔS° was estimated to be + 145 J · deg1−. mol−1. The results are consistent with an entropy‐driven reaction where polar groups establish contact surface complementarity and the specificity of the association, and hydrophobic bonding stabilises the association complex. Intact cells incubated with 59Fe‐labelled and 125I‐labelled transferrin showed a much greater dependence on incubation temperature than the binding of transferrin to its solubilized receptor. Arrhenius plots for the rate of transferrin uptake; the rate of iron uptake and rate of iron incorporation into haem all had similar ‘activation energies’. These plots exhibited ‘break points’ or discontinuities at 20°C and 45°C which were not seen with the solubilized receptor. Such ‘break points’ imply phase transitions in the reticulocyte membrane at these temperatures. The results in conjunction with those previously reported indicate that once membrane receptor occupation by transferrin has occurred the reaction which limits iron acquisition by reticulocytes is the endocytotic intracellular availability of the transferrin‐receptor complex.