Physiological levels of binding and iron donation by complementary half-molecules of ovotransferrin to transferrin receptors of chick reticulocytes

Two fragments, each corresponding to approximately half of the ovotransferrin (OTf) molecule and containing an iron-binding site were produced by digestion with affinity bound trypsin and were purified by isoelectric focusing and gel filtration chromatography. The immunologically distinct "half-molecules" individually have little ability to bind to transferrin receptors on chick embryo red blood cells or to donate iron to them. Combining them, however, leads to both binding and iron donation approaching that found for holo-OTf. Furthermore, similar amounts of radiolabeled iron can be extracted into the putative heme fraction from Fe2OTf and from the various combined half-molecules. These findings conflict with those reported by Keung and Azari ( (1982) J. Biol. Chem. 257, 1184-1188) for subtilisin-derived half-molecules of OTf examined in a similar system. They found that each half-molecule appeared to bind at a level of approximately one-third that of Fe2OTf and that the half-molecules competed with each other for binding sites. In contrast, our equilibrium binding studies, in the presence of 2,4-dinitrophenol to prevent iron removal, led to the determination of 4.79 X 10(4) binding sites/cell for Fe2OTf, 4.44 X 10(4) for the NH2-terminal half-molecules in the presence of excess COOH-terminal half-molecules and 4.17 X 10(4) for COOH-terminal half-molecules in the presence of NH2-terminal half-molecules; apparent binding constants were estimated to be 3.29 X 10(6), 1.19 X 10(6), and 0.67 X 10(6) M-1 for these same samples. Problems associated with equilibrium binding studies in which a narrow range of concentrations of ligand is used and/or iron is being removed are discussed. Labeled combined half-molecules were half as effective as labeled Fe2OTf in competition with unlabeled Fe2OTf. These findings are consistent with the lower apparent binding constant found in the equilibrium binding studies. Equimolar apo-OTf had no effect on binding of either Fe2OTf or the combined half-molecules. It seems apparent from our studies that the NH2- and COOH-terminal half-molecules each contain a recognition region both of which are necessary for binding to the transferrin receptor and iron donation to the chick embryo red blood cell.     

Binding and iron delivering of monoferric ovotransferrins to chick-embryo red blood cells (CERBC)

1. Both monoferric forms of OTf, each of about 80 kDa, bound to CERBC enough tightly, but at a lesser extent with respect to Fe2OTf with a Bmax in the order: 59Fe2OTf greater than OTf59FeC much greater than 59FeNOTf. 2. Fe2OTf competed, in equimolar ratio, with 59FeNOTf or OTf59FeC, lowering the Bmax value; a 10-fold molar excess of Fe2OTf almost abolished the binding of both labelled monoferric forms. Apo-OTf did not compete with the monoferric forms for binding to CERBC. Iron-saturated N- or C-terminal OTf half-molecules, each of about 40 kDa, were unable to displace the monoferric form. By contrast, the mixture of both half-molecules gave results very similar to Fe2OTf. 59FeNOTf and OTf59FeC were displaced by a molar excess of both unlabelled monoferric forms. 3. Uptake experiments showed that both monoferric forms of OTf were less effective in delivering iron to CERBC with respect to the diferric form, but, nevertheless, there was still an appreciable iron uptake which paralleled the binding behaviour, being the C-form slightly more efficient than the N-form.     

Differential effect of iodination of ovotransferrin and its two half-molecule domains on binding to transferrin receptors on chick embryo red blood cells.

Iodination of the C-terminal half-molecule domain of ovotransferrin (OTF) causes a significant reduction in binding to transferrin receptors on chick reticulocytes when compared to the binding observed with holo-OTF or the N-terminal half-molecule domain. (In such studies binding of iodinated half-molecule is measured in the presence of equimolar unlabelled complementary half-molecule). In particular iodination of the C-terminal half-molecule domain by the solid-phase reagent Iodogen resulted in half the binding found when ICl was used. The iodinated N-terminal half-molecule domain labelled by either Iodogen or ICl showed consistently higher binding than was observed with the C-terminal half-molecule or Fe2OTF. Although the molecular basis for the reduced binding of these proteins relative to the N-terminal half-molecule has not been definitively established, the implication is that there is a Tyr in the C-terminal domain which is involved in receptor recognition and binding. Addition of one or more bulky iodine atoms to the Tyr interferes with the interaction. Tryptic peptide maps of unlabelled holo-OTF and half-molecule domains and of the half-molecule domains labelled by both ICl and Iodogen are presented. The maps indicate limited access of the tyrosine residues to iodination especially in the C-terminal half-molecule domain. Equilibrium binding experiments have been carried out to compare the Kd (the apparent dissociation constant for the interaction between OTF and the transferrin receptors on chick-embryo red blood cells) with the Bmax, (binding at infinite free-ligand concentration) for Fe2OTF labelled using ICl, Iodogen, Enzymobeads and Chloramine-T. The effect of labelling Fe2OTF by Bolton-Hunter reagent has also been assessed. These studies show that ICl appears to be the reagent of choice for labelling Fe2OTF and its half-molecule domains.     

The soluble form of the membrane-bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue found particularly in melanoma cells. Apart from membrane-bound MTf, a soluble form of the molecule (sMTf) has been identified in vitro[Food, M.R., Rothenberger, S., Gabathuler, R., Haidl, I.D., Reid, G. & Jefferies, W.A. (1994) J. Biol. Chem.269, 3034-3040] and in vivo in Alzheimer's disease. However, nothing is known about the function of sMTf or its role in Fe uptake. In this study, sMTf labelled with 59Fe and 125I was used to examine its ability to donate 59Fe to SK-Mel-28 melanoma cells and other cell types. sMTf donated 59Fe to cells at 14% of the rate of Tf. Analysis of sMTf binding showed that unlike Tf, sMTf did not bind to a saturable Tf-binding site. Studies with Chinese hamster ovary cells with and without specific Tf receptors showed that unlike Tf, sMTf did not donate its 59Fe via these pathways. This was confirmed by experiments using lysosomotropic agents that markedly reduced 59Fe uptake from Tf, but had far less effect on 59Fe uptake from sMTf. In addition, an excess of 56Fe-labelled Tf or sMTf had no effect on 125I-labelled sMTf uptake, suggesting a nonspecific interaction of sMTf with cells. Protein-free 125I determinations demonstrated that in contrast with Tf, sMTf was markedly degraded. We suggest that unlike the binding of Tf to specific receptors, sMTf was donating Fe to cells via an inefficient mechanism involving nonspecific internalization and subsequent degradation.     

Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites.

The transferrin receptor (TfR) binds two proteins critical for iron metabolism: transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. Previous results demonstrated that Tf and HFE compete for binding to TfR, suggesting that Tf and HFE bind to the same or an overlapping site on TfR. TfR is a homodimer that binds one Tf per polypeptide chain (2:2, TfR/Tf stoichiometry), whereas both 2:1 and 2:2 TfR/HFE stoichiometries have been observed. In order to more fully characterize the interaction between HFE and TfR, we determined the binding stoichiometry using equilibrium gel-filtration and analytical ultracentrifugation. Both techniques indicate that a 2:2 TfR/HFE complex can form at submicromolar concentrations in solution, consistent with the hypothesis that HFE competes for Tf binding to TfR by blocking the Tf binding site rather than by exerting an allosteric effect. To determine whether the Tf and HFE binding sites on TfR overlap, residues at the HFE binding site on TfR were identified from the 2.8 A resolution HFE-TfR co-crystal structure, then mutated and tested for their effects on HFE and Tf binding. The binding affinities of soluble TfR mutants for HFE and Tf were determined using a surface plasmon resonance assay. Substitutions of five TfR residues at the HFE binding site (L619A, R629A, Y643A, G647A and F650Q) resulted in significant reductions in Tf binding affinity. The findings that both HFE and Tf form 2:2 complexes with TfR and that mutations at the HFE binding site affect Tf binding support a model in which HFE and Tf compete for overlapping binding sites on TfR.     

Simultaneous multiple element detection by particle beam/hollow cathode-optical emission spectroscopy as a tool for metallomic studies: determinations of metal binding with apo-transferrin

Particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) is presented as a tool for the determination of metal ion loading in transferrin (Tf). The elemental specificity of optical emission spectroscopy provides a means of assessing metal ion concentrations as well as the relative amounts of metal per unit protein concentration (up to 2 moles of Fe per mole of protein). The PB/HC-OES method allows for the simultaneous detection of metal content (Fe (I) 371.99, Ni (I) 341.41 nm, Zn (I) 213.86 nm, and Ag (I) 338.28 nm in this case), as well as elemental carbon and sulfur (C (I) 156.14 nm and S (I) 180.73 nm) that are reflective of the protein composition and concentration. Quantification for the metal species is based on calibration functions derived from aqueous solutions, with limits of detection for the entire suite being less than 1.0 μM. Determinations in this manner eliminate much of the ambiguity inherent in UV-VIS absorbance determinations of Tf metal binding. Validation of this method is obtained by analyzing loading response of Fe(3+) into Tf using the PB/HC-OES method and comparing the results with those of the standard UV-VIS absorbance method. Maximum Fe(3+) loading of Tf (based on the number of available binding sites) was determined to be 71.2 ± 4.7% by the PB/HC-OES method and 67.5 ± 2.5% for the UV-VIS absorbance method. Element emission ratios between the dopant metals and the carbon and sulfur protein constituents allow for concentration independent determinations of metal binding into Tf. Loading percentages were determined for Ni(2+), Zn(2+), and Ag(+) into Tf with maximum loading values of 19.5 ± 0.4%, 41.0 ± 4.4%, and 141.2 ± 4.3%, respectively. While of no apparent biological significance, Ag(+) presents an interesting case as a surrogate for Pt(2+), whose binding with Tf has shown to be quite different from the other metals. A different mode from the others is indeed observed, and is consistent with conjecture on the Pt(2+) mechanisms. Competitive binding studies not easily performed using absorbance spectroscopy are easily performed by simultaneous, multielement analysis, reflective of the metals and the protein content. In this work, there is clear competition between and Fe(3+) and Zn(2+) for binding in the C-terminus lobe of Tf, while Ni(2+) binds within the N-terminus lobe. Addition of Ag(+) to this mixture does not affect the other metals' distributions, but reflects binding at other protein sites.     

Light-scattering investigation of the subunit structure and dissociation of Helix pomatia hemocyanin. Effects of salts and ureas

The effects of various salts of the Hofmeister and aliphatic acid salt series and hydrophobic reagents of the urea series on the subunit structure and the dissociation of Helix pomatia alpha-hemocyanin were investigated by employing light-scattering molecular weight methods. In moderate ranges of salt concentrations [0-1.0 M NaClO4, NaSCN, NaI, and guanidinium chloride (GdmCl) and 0-2.0 M NaBr], the dissociation reaction is essentially a two-step process characterized by the dissociation of whole hemocyanin molecules dissociating to half-molecules of decamers followed by the dissociation of the half-molecules to five dimeric fragments. The effectiveness of the salts and relative ineffectiveness of the ureas and GdmCl as dissociating agents in the first step of the dissociation reaction suggest that the stabilization of the contact areas between half-molecules in solution is largely a nonhydrophobic energy process involving polar and ionic interactions. Hydrophobic forces appear to be important, however, for stabilization of the half-molecules through side to side contacts of the five dimeric units that make up each half-molecule. The analysis of our dissociation data by use of equations derived in our previous studies [Herskovits, T. T., & Harrington, J.P. (1975) Biochemistry 14, 4964-4971] gave apparent estimates of amino acid groups of about 60-150 for each of the contact areas between the cylindrically shaped half-molecules and 30-60 for each of the dimers in the half-molecules themselves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of transferrin in metal uptake by human lymphoblasts in vitro

The uptake and binding of 59Fe, 67Ga and 239Pu complexed with citrate of transferrin (Tf) and of 125I-labelled Fe-Tf by human lymphoblasts (WI-L2 cells) have been studied. Uptake kinetics of 59Fe-Tf and [125I]-Tf point to internalization by receptor mediated endocytosis. 67Ga binding and uptake is always less. This may be explained by a lower affinity of Ga-complexes for the cell surface. Factors which influence Fe uptake have a similar effect on Ga. 239Pu uptake and binding, however, are different, especially in that Tf does not stimulate 239Pu uptake and may actually decrease it.     

Subunit dissociation in the allosteric regulation of glycerol kinase from Escherichia coli. 2. Physical evidence.

The dependence of the molecular weight of glycerol kinase on enzyme concentration and on binding of fructose 1,6-bisphosphate has been examined by velocity sedimentation, gel filtration, and polyacrylamide gel electrophoresis. The sedimentation coefficient and Stokes radius decrease as a consequence of dilution in a manner consistent with dissociation into half-molecules, with limiting values suggesting molecular weights of about 218,000 and 136,000 for the associated and dissociated species, respectively. Fructose 1,6-bisphosphate (5 mM) prevents the decrease in sedimentation coefficient brought about by dilution, suggesting a decrease in the apparent subunit dissociation constant of at least four orders of magnitude. Electrophoretic mobility in polyacrylamide gels increases as a consequence of dilution in the absence, but not in the presence, of fructose 1,6-bisphosphate. Ferguson plots indicate that glycerol kinase has the same molecular weight in the presence of fructose 1,6-bisphosphate as the covalently cross-linked tetramer and is substantially smaller in the absence of fructose 1,6-bisphosphate. These results are consistent with the model of glycerol kinase proposed in the preceding paper of this issue [de Riel, J.K., and Paulus, H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding.     

Differed preferential iron-binding lobe in human transferrin depending on the presence of bicarbonate detected by HPLC/high-resolution inductively coupled plasma mass spectrometry

The binding of iron (Fe) to human serum transferrin (Tf) was analyzed with an HPLC system equipped with an anion exchange column and directly connected with a high-resolution inductively coupled plasma mass spectrometer for metal detection. The (56)Fe level in the eluate was monitored at resolution m/Deltam=3000. Two monoferric Tfs were assigned based on the results of urea-PAGE and desferrioxamine experiments. When Fe was added as Fe-citrate stepwise to an apo-Tf solution in the presence of bicarbonate, the N-lobe site was the preferential Fe-binding site, while the C-lobe site was preferred in the absence of bicarbonate. In both cases, the Fe-peak areas of the preferential site and Fe(2)-Tf increased up to an Fe/Tf molar ratio of 1, and then the peak area of the monoferric Tf decreased while the peak area of Fe(2)-Tf increased. When the Fe/Tf molar ratio was below 1, the amount of Fe bound to the lobe with a weaker affinity was higher in Fe(2)-Tf than in the monoferric Tf in each case. Namely, Fe(2)-Tf was the preferential binding state of Fe to human serum Tf. The preference is reasonable for transferring Fe ions effectively to Tf-receptors.     

Neither human hephaestin nor ceruloplasmin forms a stable complex with transferrin

Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Cell toxicity is caused by the generation of hydroxyl-free radicals that result from redox reactions involving Fe(II). Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of these harmful by-products. Ceruloplasmin (Cp) is the major multicopper ferroxidase in blood; however, hephaestin (Hp), a membrane-bound Cp homolog, was recently discovered and has been implicated in the export of iron from duodenal enterocytes into blood. In the intracellular milieu, it is likely that iron exists as reduced Fe(II), yet transferrin (Tf), the plasma iron transporter, is only capable of binding oxidized Fe(III). Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between a ferroxidase and the iron transporter. As such, it has been suggested that as a means of preventing the release of unbound Fe(III), a direct protein-protein interaction may occur between Tf and Hp during intestinal iron export. In the present study, the putative interaction between Tf and both Cp and a soluble form of recombinant human Hp was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance (SPR), a stable interaction between the two proteins was not detected. We conclude that a stable complex between these ferroxidases and Tf does not occur under the experimental conditions used. We suggest alternative models for loading Tf with Fe(III) during intestinal iron export.     

Preparation of iron-deficient tissue culture medium by deferoxamine-sepharose treatment and application to the differential actions of apotransferrin and diferric transferrin.

We have shown that triiodothyronine-dependent GH1 rat pituitary cell growth in serum-free defined culture required apotransferrin (apoTf) (D. A. Sirbasku, et al., Biochemistry 30, 295-304, 7466-7477, 1991). These studies were done in "low-Fe" medium without Fe(III)/Fe(II) salts. Nonetheless, significant concentrations of iron may have been contributed by other components, making this medium unsuitable for study of the differential effects of apoTf and diferric transferrin (2Fe.Tf). Measuring residual iron in culture medium has been troublesome because the most sensitive method (i.e., atomic absorption) detected levels only in excess of 10 ng/ml and did not distinguish between the forms of iron present. To estimate the Fe(III) available to bind to apoTf, we developed a more sensitive and specific method. Urea-polyacrylamide gel electrophoresis (PAGE) separates apoTf, the two monoferric transferrins, and 2Fe.Tf. [125I]apoTf was incubated with medium, or components, and the formation of [125I]-2Fe.Tf was monitored by urea-PAGE/autoradiography. By this method, the concentration of Fe(III) in low-Fe medium was estimated at 8.4 to 20 ng/ml and the sources were identified. We next sought to remove the Fe(III). Standard chelators were ineffective or cytotoxic. In contrast, an affinity method with deferoxamine-Sepharose depleted greater than or equal to 90% of the Fe(III). In this medium, apoTf and 2Fe.Tf showed differential effects with GH1 cells and with MCF-7, MTW9/PL2, an MDCK cells. With the methods described here, the effects of apoTf and 2Fe.Tf on growth can be studied separately.     

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.     

Aluminum exposure affects transferrin-dependent and -independent iron uptake by K562 cells

Aluminum (Al) and iron (Fe) share several physicochemical characteristics and they both bind to transferrin (Tf), entering the cell via Tf receptors (TfR). Previously, we found similar values of affinity constant for the binding of TfR to Tf carrying either Al or Fe. The competitive interaction between both metals prevented normal Fe incorporation into K562 cells and triggered the upregulation of Fe transport. In the present work we demonstrated that Al modified Fe uptake without affecting the expression of Tf receptors. Both TfR and TfR2 mRNA levels, evaluated by RT-PCR, and TfR antigenic sites, analyzed by flow cytometry, were found unchanged after Al exposure. In turn, Al did induce upregulation of non-Tf bound Fe (NTBI) uptake. This modulation was not due to intracellular Fe decrease since NTBI transport proved not to be regulated by Fe depletion. Unlike its behavior in the presence of Tf, Al was unable to compete with NTBI uptake, suggesting that both metals do not share the same alternative transport pathway. We propose that Al interference with TfR-mediated Fe incorporation might trigger the upregulation of NTBI uptake, an adaptation aimed at incorporating the essential metal required for cellular metabolism without allowing the simultaneous access of a potentially toxic metal.     

Physical and chemical studies on bacterial superoxide dismutases. Purification and some anion binding properties of the iron-containing protein of Escherichia coli B.

Highly purified iron superoxide dismutase was obtained from Escherichia coli B using a modification of the procedure of Yost and Jridovich (Yost, F. J., Jr., and Fridovich, I. (1973) J. Biol. Chem. 248, 4905-4908). The protein contained 1.8 +/- 0.2 atoms of iron per 38,700 g of protein. We have found that cyanide does not bind to the Fe3+ ion of iron dismutase but fluoride and azide have moderately large binding constants. Optical and electron paramagnetic resonance (EPR) measurements suggested that 2 fluoride ions could associate with each iron atom with the first having an association constant of approximately 520 M-1 and the second with an estimated value of 24 M-1. Activity measurements yielded an inhibition constant for fluoride of 30 M-1. At room temperature only one azide binds to the Fe3+ (K = 760 M-1) and this does not interfere with superoxide dismutase activity. Upon freezing solutions of iron superoxide dismutase in the presence of excess azide their color changes from yellow to pink. Combined EPR and optical titrations with azide suggest the presence of two binding sites on Fe3+ with only the first being occupied at room temperature and the second binding azide only upon freezing the solution. The results suggest that each Fe3+ ion of this superoxide dismutase has two coordination positions available for interaction with solute molecules but only one is necessary for catalysis of the superoxide dismutation reaction. The EPR, optical, and circular dichroism spectra of the native protein and the various fluoride and azide complexes are presented.     

Comparative studies of the binding and growth-supportive ability of mammalian transferrins in human cells

The ability of human-derived cells in culture to bind, remove iron from, and grow in the presence of transferrins (Tf) isolated from the sera of species commonly included in tissue culture medium was investigated. Kinetic studies on HeLa cells reveal apparent first-order association rate constants of 0.43 min-1 for human Tf and 0.15 min-1 for equine Tf. Labeled chicken ovo-Tf and fetal bovine Tf were not recognized by the HeLa cells. Competition experiments with HeLa cells that use either isolated Tf or parent serum confirm these findings. Equilibrium binding experiments performed on HeLa cells at 37 degrees C in the presence of 2,4-dinitrophenol to prevent iron removal indicate 1 X 10(6) Tf bound/cell with a dissociation constant (K'D) of 28 nM for human Tf and 182 nM for equine Tf. Equilibrium binding performed at 0 degrees C to prevent endocytosis reveals 4.1-6.7 X 10(5) Tf binding sites/cell with a K'D of 8.3 nM for human Tf and 41.5 nM for equine Tf. Parallel experiments in normal human diploid fibroblast-like MRC-5 cells indicate expression of 0.82-2.78 X 10(5) Tf binding sites/cell with a K'D of 8.2 nM for human and 39.1 nM for equine Tf. Thus, the results of equilibrium binding studies of a more differentiated cell type are consistent with those found for HeLa cells. Fetal bovine Tf was found to compete weakly with labeled human Tf for human receptor on HeLa cells in a soluble receptor assay, with an approximately 500-fold excess needed to reduce binding to half maximal. Iron uptake experiments show an iron donating hierarchy where human greater than horse greater than calf, suggesting that the rate of iron uptake depends on the affinity of receptor for transferrin. Growth experiments involving HeLa cells in chemically defined serum-free medium demonstrate that bovine Tf will support growth as well as human Tf, but at concentrations much higher than are required of human Tf.     

Specificity of siderophore receptors in membrane vesicles of Bacillus megaterium.

Membrane vesicles of Bacillus megaterium strains SK11 and Ard1 bound the ferrischizokinen and ferriferrioxamine B siderhores (iron transport cofactors). An approximately equimolar uptake of both labels of [3H, 59Fe]ferrischizokinen indicated binding of the intact chelate. Binding reached equilibrium in 2 to 5 min, was temperature independent, and was unaltered by the addition of several energy sources. A 91% dissociation of bound [Fe]ferrischizokinen was achieved in 60 s by the addition of excess ferrischizokinen. Ferriaerobactin, a siderophore which is structurally related to ferrischizokinen, caused no detectable release of bound [59Fe]ferrischizokinen. Of several other ferrigydroxamates tested, only ferriferrichrome A achieved the release (11%) of [Fe]ferrischizokinen. Rapid dissociation (92%) of bound [59Fe]ferriferrioxamine B by the addition of ferriferrioxamine B was observed, and a 67% release of [59Fe]ferriferrioxamine B was caused by ferriA2265, its structural relative. Ferrischizokinen, ferriferrichrome A, and ferrirhodotorulic acid produced a 6, 25, and 29% dissociation, respectively, of [59Fe]ferriferrioxamine B; ferriaerobactin caused no dissociation. [59Fe]ferriaerobactin was bound by the membranes, but its dissociation was not effected by unlabeled ferriaerobactin, suggesting no specific receptors for this chelate. The respective binding affinity constants and maximal binding capacities of membrane vesicles of strain SK11 were 2 x 10(7) M-1 and 280 pmol per mg of protein for ferrischizokinen and 7 x 10(7) M-1 and 37 pmol per mg of protein for ferriferrioxamine B. These values in strain Ard1 were, respectively, 1.4 x 10(7) M-1 and 186 pmol per mg of protein for ferrischizokinen and 11 x 10(7) M-1 and 23 pmol per mg of protein for ferriferrioxamine B. Separate, specific binding sites (receptors) for ferrischizokinen and ferriferrioxamine B exist on the vesicles. The ferrischizokinen receptors have a lower affinity but a higher binding capacity (eightfold) than that shown by the ferriferrioxamine B receptor. These receptors may be components of independent transport systems.     

Regulation of interleukin-1 receptors on AtT-20 mouse pituitary tumour cells.

To study the cellular mechanisms of interleukin-1 (IL-1) in the pituitary corticotroph, we studied the properties of IL-1 receptors on a mouse pituitary ACTH-producing cell line, AtT-20. Scatchard plot analysis revealed a single type of receptor with a Kd (dissociation constant) of 93 pM, and 482 binding sites/cell. [125I]IL-1 alpha binding competed with IL-1 alpha and IL-1 beta in an equimolar fashion. A 24 h pre-incubation with either CRH, epinephrine or nor-epinephrine increased the [125I]IL-1 alpha binding sites in the AtT-20 cells and conversely, a similar pre-incubation with either dexamethasone or tumour necrosis factor-alpha (TNF alpha) decreased them without affecting the affinity of the receptors in either case.     

Titanium preferential binding sites in human serum transferrin at physiological concentrations

Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants.     

Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.