Iron uptake from rat plasma transferrin by rat reticulocytes.

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Antigenic material in rat plasma reacting with rat transferrin receptor antibodies was identified as an intact receptor molecule complexed with transferrin. Plasma transferrin receptors were measured by ELISA in rats of different age and sex, of different iron status, with different degrees of erythropoiesis, and with inflammation. An inverse relationship between iron status and receptor number was found, whereas a direct relationship existed between erythropoiesis and receptors. These changes in receptor number can be explained by assuming that the number of tissue receptors determined the number of plasma receptors and that the erythroid cells possessed most of the body's receptors. Increases in plasma receptors lagged behind the appearance of circulating reticulocytes, suggesting that receptors were released to the plasma during the terminal phase of erythrocyte maturation.Iron-bearing transferrin in the plasma interacts with specific membrane receptors to deliver iron to cells (1). The process involves the internalization of the complex in a vesicle, the liberation of the iron to the cell's cytoplasm, and the return of the receptor to the cell membrane and of transferrin to the plasma (2). It is well documented in cell culture that the number of cell receptors bears an inverse relationship to the cell's iron status (3). Most of the in vivo transferrin-mediated iron uptake is by the erythroid marrow, so that tissue is assumed to contain most of the functional transferrin receptors and changes in erythropoiesis would affect receptor number. Studies by Kohgo et al. (4) have demonstrated material in human plasma, presumably transferrin receptor, that reacts with receptor antibodies. Kohgo has also shown increases in the amount of plasma receptor material in patients with hemolytic anemia and iron deficiency (5). In this study, we describe the presence in rat plasma of a receptortransferrin complex, reacting with either receptor or transferrin antibody. Results of a quantitative assay of plasma receptor levels under a variety of conditions are reported. METHODSReceptor Characterization Procedures. Electrophoresis was carried out in an electrophoretic cell model 155 (Bio-Rad, Richmond, CA), using 7.5% polyacrylamide gels in a column of 1.4 x 12 cm. 59Fe-labeled rat plasma samples (0.3 ml) were subjected to electrophoresis for 20 hr at 80 V. Thereafter, the gel was cut in slices of 2-mm thickness. The content of each slice was eluted for 48 hr in 1 ml of 0.9o saline containing 0.4% TERIC, and analyzed for receptor content by ELISA. 59Fe was counted in a Packard model 5330 y counter (Packard Instruments).Anti-transferrin antibodies were produced after immunization of rabbits as described (6, 7). Crossed immunoelectrophoresis was carried out by standard procedures (7).Quantitative Measurement of Transferrin Receptors. Antibody production was undertaken by using rat placental receptor-transferrin complexes isolated as described elsewhere (1). The complex had the advantage of possessing greater stability tha...

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Transferrin receptors in rat plasma.

Béguin

Huebers

Josephson

et al. 1988

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“…There has been considerable discussion concerning the behavior of this protein in view of the chemical differences between its two iron binding sites (1). Our recent studies, however, have shown that iron loading of the binding sites is essentially a random process and the release ofiron on incubation with erythroid cells is an "all-or-none" phenomenon (2)(3)(4)(5). In addition, it was shown that iron delivery from the two monoferric transferrins is identical when they are incubated in vitro with reticulocytes or bone marrow cells (4,6) or injected in vivo into rabbits (7).…”

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Competitive advantage of diferric transferrin in delivering iron to reticulocytes.

Huebers¹,

Csiba²,

Huebers³

et al. 1983

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Radioiron-and radioiodine-labeled forms of human diferric and monoferric transferrin and apotransferrin, isolated by preparative isoelectric focusing, were used to define transferrin-iron uptake by human reticulocytes. In mixtures of human diferric and monoferric transferrin, the diferric molecule had a constant 7-fold advantage in delivering iron to reticulocytes, as compared with the 2-fold advantage when single solutions of mono-and diferric transferrins were compared. This was shown to be due to competitive interaction in iron delivery, probably at a common membrane-receptor binding site for transferrin. Apotransferrin did not interfere with the iron-donating process and its limited cellular uptake was inhibited in noncompetitive fashion by diferric transferrin.Transferrin mediates iron exchange between body tissues. There has been considerable discussion concerning the behavior of this protein in view of the chemical differences between its two iron binding sites (1). Our recent studies, however, have shown that iron loading of the binding sites is essentially a random process and the release ofiron on incubation with erythroid cells is an "all-or-none" phenomenon (2-5). In addition, it was shown that iron delivery from the two monoferric transferrins is identical when they are incubated in vitro with reticulocytes or bone marrow cells (4,6) or injected in vivo into rabbits (7). In all these experiments, diferric transferrin proved to be a better donor than the single-iron-loaded molecule (2-5, 8), in variation with an earlier report (9). While part of this advantage relates to the simultaneous release of both iron atoms, an additional preference for the diferric form has been suggested but not quantitated (10). In this report, the relationship ofiron supplied to human reticulocytes from human mono-and diferric transferrin is examined at physiological levels of plasma iron. In addition, the effect of apotransferrin on the iron delivery process was studied. MATERIALS AND METHODS Transferrin Preparations. Purification and radioiodinationof human diferric transferrin by using "1I and 131I were done as described (3,4). Conversion of the transferrin into apotransferrin was accomplished by using desferrioxamine (4).For the preparation of di[mFe]ferric transferrin, 90 ,uCi of 55Fe (10,Ci/,ugas FeSO 1 Ci = 37GBq; NewEngland Nuclear) was added to 200 mg of t'I-labeled apotransferrin (100 ,uCi of "'lI dissolved in 5 ml of 0.05 M Tris-HCl, pH 8.0/0.01 M NaHCO3) and this was followed by the addition ofsufficient iron as ferrous ammonium sulfate (39 ,g of iron/ml in 0.01 M HCl) to exactly saturate the free iron binding sites present in the transferrin solution. * This goal was achieved by spectrophotometric titration techniques in which the increase in color development at A = 465 nm was observed (11). At the point of saturation, the A465/Am ratio of the transferrin solution was 0.045, which is characteristic of the diferric state of the transferrin molecule (2, 3). Transferrin solutions were prepared at iron s...

“…The two separable and chemically distinct monoferric forms of transferrin (AS and AL) have been shown to have identical iron-donating capacities, both in animals and in humans (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). In addition, iron release from any form of transferrin to erythroid cells was found to be an "all or none" phenomenon, generating only apotransferrin (5)(6)(7)(8)(9)(10)(11).…”

Section: Resultsmentioning

confidence: 99%

“…A heterogeneous behavior of the plasma iron pool could lead to a selective release of iron from one site, a selective loading of the two sites, or a combination of these. Recent studies failed to reveal functional differences in iron release between the two sites (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Instead, a competitive advantage of difemc transferrin over the two monoferric forms in delivering iron was apparent (13,14).…”

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confidence: 99%

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Occupancy of the iron binding sites of human transferrin.

Huebers¹,

Josephson²,

Huebers³

et al. 1984

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The in vivo distribution of iron between the binding sites of transferrin was examined. Plasma was obtained from normal subjects under basal conditions and after in vitro and in vivo iron loading. Independent methods, including measurement of the transferrin profile after isoelectric focusing and cross immunoelectrophoresis, and determination of the iron content in the separated fractions were in agreement that there was a random distribution of iron on binding sites. This held true with in vitro loading, when iron was increased by intestinal absorption and with loading from the reticuloendothelial system. The MATERIALS AND METHODSFresh heparinized plasma samples were obtained from normal adults after an overnight fast. Loading of plasma with iron above basal levels was achieved in vitro by adding ferrous sulfate (4, 6) and in vivo either through intestinal absorption after oral ingestion of 100 mg of iron as ferrous ascorbate (molar ratio Fe/ascorbate = 1:5) or from the reticuloendothelial system through hemolysis induced by oral ingestion of 300 mg of niacin (15, 16). In both in vivo loading studies, plasma samples were obtained at basal levels of saturation and at 20-min intervals for up to 6 hr after drug ingestion. These plasma samples were trace-labeled by the addition of 125I-labeled apotransferrin or with 59Fe.Labeling of transferrin with 1251 was carried out by the iodine monochloride procedure of McFarlane (17) with modifications as described (5, 6). To 4 ml of the plasma to be analyzed was added 0.2 ,uCi (1 Ci = 37 GBq) of 125I-apotransferrmn (specific activity, 0.4 mCi/mg of protein). The 59Fe was added as ferrous sulfate (0.1 ,uCi in 0.1 ml of 0.01 M HCl (specific activity, 10 ,uCi/,tg of Fe), to the same 4 ml of plasma. The mixture was incubated for 10 min at 370C and thereafter stored at 40C.Electrophoresis in the presence of 6 M urea was based on the separation principle described by Makey and Seal (18), and crossed immunoelectrophoresis was based on the procedures described by Weeke and Soderholm et al. (19,20). The replacement of polyacrylamide by Sea Kem LE agarose (FMC, Marine Colloid Division, Rockland, ME) gave better separation and required less manipulation than did the system described by Leibman and Aisen (21). This particular type of agarose will gel even in the presence of 6 M urea. Reagents were prepared with deionized water and further treated with a chelating resin (Chelex 100, Bio-Rad) to remove any residual contaminating iron (22). The 10 M urea stock solution was mixed for 5 hr with bed resin (50 g/liter; AG501-X8, Bio-Rad), filtered, and stored at 40C. The barbital buffer (0.1 M, pH 8.6) was mixed (5 g/liter) with Chelex 100 and then filtered. In the actual procedure, a mixture of 45 ml of 10 M urea, 35 ml of 0.1 M barbital buffer, and 0.75 g of Sea Kem LE agarose was heated to boiling. We poured the gel in a cold room (2°C) using a precooled leveling table in a humidity chamber. A volume of 16.5 ml of gel per 110 x 102.5 mm sheet of agarose gel support medium (Gel B...