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1.
Edward S. Cole Jonathan Glass 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,762(1):102-110
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 1.14·105molecules/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 4.4·104±1.9·104 for cells in suspension and 6.6·104±2.3·104 for adherent cultured cells. The apparent association constants were 1.23·107 1·mol?1 and 3.4·106 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 Fe11 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. 相似文献
2.
Iron uptake from transferrin and transferrin endocytic cycle in Friend erythroleukemia cells 总被引:2,自引:0,他引:2
Several aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of hemoglobin synthesis by dimethyl sulfoxide. The maximal rate of iron uptake from 59Fe-labeled transferrin, 1.5 X 10(6) atoms of Fe/cell per 30 min in uninduced cells, increased to 3 X 10(6) atoms/cell after 5 days of induction. The increase in iron uptake was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I-labeled transferrin binding, suggesting a more efficient iron uptake by transferrin receptors in induced cells, with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this conclusion are results of the study of cellular 125I or 59Fe labeled transferrin kinetics. In the induced cells transferrin endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. On the other hand, transferrin release by uninduced cells was significantly slower and a substantial part of internalized 59Fe was released. On the basis of these results, different efficiency of iron release from internalized transferrin, accompanied by changes in cellular transferrin kinetics, is proposed as one of the factors determining the rate of iron uptake by developing erythroid cells. 相似文献
3.
The p97 antigen is mapped to the q24-qter region of chromosome 3; the same region as the transferrin receptor. 总被引:2,自引:1,他引:1
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P A Seligman C D Butler E J Massey J A Kaur J P Brown G D Plowman Y Miller C Jones 《American journal of human genetics》1986,38(4):540-548
Since the p97 antigen, a membrane-associated iron-binding protein, has extensive amino acid sequence with homology with transferrin, is functionally related to the transferrin receptor, and has been previously mapped to chromosome 3, we have performed additional studies for regional mapping of the gene expressing p97 antigen. In these experiments, Chinese hamster-human cell lines were chosen that contained a large spectrum of autosomal human chromosomes, but mainly consisted of clones expressing all or a part of chromosome 3. These cell lines included a clone that previously allowed for mapping of human transferrin receptor to q22-qter region. Human p97 expression was assessed by specific binding of [125I]monoclonal antibody 96.5, and human transferrin receptor expression was tested by specific [125I]human transferrin binding and [125I]monoclonal antibody OKT-9 specific for human transferrin receptor. Based on these analyses, both human p97 antigenic expression and human transferrin receptor are mapped concordantly to the q24-qter region. These data and previous reports, therefore, suggest that the related iron-transport proteins are closely linked and may be under coordinate regulation. However, studies of several cell lines that exhibit up-regulation of human transferrin receptor expression with cellular proliferation, and down-regulation of receptor with increased transferrin-iron in the media, showed no change in expression of p97 antigen. p97 antigenic expression increased when melanocyte-stimulating hormone was added to a human melanoma cell line in tissue culture. These latter studies suggest that in mammalian cells the two proteins do not show coordinate regulation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Kumar S Sheokand N Mhadeshwar MA Raje CI Raje M 《The international journal of biochemistry & cell biology》2012,44(1):189-199
A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron. 相似文献
5.
Albumin prevents nonspecific transferrin binding and iron uptake by isolated hepatocytes 总被引:5,自引:0,他引:5
Bovine serum albumin inhibits binding of transferrin by hepatocytes in suspension by 60-70%. Iron uptake is inhibited by less than 20%. A Scatchard analysis of the transferrin-binding data reveals a biphasic plot in the absence of bovine serum albumin, but a monophasic plot in the presence of bovine serum albumin. Bovine serum albumin inhibits low-affinity binding of transferrin (125000 molecules/cell), but has no effect on high-affinity binding (38000 molecules/cell). In pronase-treated cells, transferrin binding is reduced by 40%, and when bovine serum albumin is added, the binding is reduced by a further 40%. Corresponding figures for iron uptake are 70 and 10%, respectively. The results are strong evidence that the major part of iron uptake by hepatocytes occurs from transferrin bound to the plasma membrane transferrin receptor. 相似文献
6.
We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin-primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate. 相似文献
7.
The release of iron and transferrin from the human melanoma cell 总被引:3,自引:0,他引:3
The role of the transferrin homologue, melanotransferrin (p97), in iron metabolism has been studied using the human melanoma cell line, SK-MEL-28, which expresses this antigen in high concentrations. The release of iron and transferrin were studied after prelabelling cells with human transferrin doubly labelled with iron-59 and iodine-125. Approx. 45% of internalised iron was in ferritin with little redistribution during reincubation. Iron release was linear with time, while transferrin release was biphasic, suggesting that iron was leaving the cell independently of transferrin. Unlabelled diferric transferrin increased transferrin release, implying a degree of coupling between cell surface binding, internalisation and release of transferrin. Increasing the preincubation time increased the amount of transferrin which remained internalised within the cell. A membrane-bound, iron-binding component with properties consistent with melanotransferrin was observed. Desferrioxamine or pyridoxal isonicotinoyl hydrazone could not remove iron from this compartment, suggesting a high affinity for iron. The number of membrane iron-binding molecules per cell was estimated to be 387,000 +/- 7000 . The non-transferrin-bound membrane Fe did not decrease during reincubation periods up to 5 h, suggesting that the cell was not utilising it. Hence, melanotransferrin may not have a role in internalising iron in melanoma cells. 相似文献
8.
Vera Neumannova Des R. Richardson Karin Kriegerbeckova Jan Kovar 《In vitro cellular & developmental biology. Animal》1995,31(8):625-632
Summary Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake,
mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates
(Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low
molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either
transferrin (5 μg/ml) or a higher concentration of ferric citrate (500 μM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth
in transferrin-free medium with a substantially lower concentration of ferric citrate (5 μM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron.
Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically
blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in
transferrin-free, low-iron medium (5 μM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was
observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine,
inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a
previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations
of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the
well-characterized receptor-mediated endocytosis process. 相似文献
9.
S Taketani H Kohno T Sawamura R Tokunaga 《The Journal of biological chemistry》1990,265(23):13981-13985
To investigate the regulation mechanism of the uptake of iron and heme iron by the cells and intracellular utilization of iron, we examined the interaction between iron uptake from transferrin and hemopexin-mediated uptake of heme by human leukemic U937 cells or HeLa cells. U937 cells exhibited about 40,000 hemopexin receptors/cell with a dissociation constant (Kd) of 1 nM. Heme bound in hemopexin was taken up by U937 cells or HeLa cells in a receptor-mediated manner. Treatment of both species of cells with hemopexin led to a rapid decrease in iron uptake from transferrin in a hemopexin dose-dependent manner, and the decrease seen in case of treatment with hemin was less than that seen with hemopexin. The decrease of iron uptake by hemopexin contributed to a decrease in cell surface transferrin receptors on hemopexin-treated cells. Immunoblot analysis of the transferrin receptors revealed that the cellular level of receptors in U937 cells did not vary during an 8-h incubation with hemopexin although the number of surface receptors as well as iron uptake decreased within the 2-h incubation. After 4 h of incubation of the cells with hemopexin, a decrease of the synthesis of the receptors occurred. Thus, the down-regulation of transferrin receptors by hemopexin can be attributed to at least two mechanisms. One is a rapid redistribution of the surface receptor into the interior of the cells, and the other is a decrease in the biosynthesis of the receptor. 59Fe from the internalized heme rapidly appeared in non-heme iron (ferritin) coincidently with the induction of heme oxygenase. The results suggest that iron released from heme down-regulates the expression of the transferrin receptors and iron uptake. 相似文献
10.
Aisen P 《The international journal of biochemistry & cell biology》2004,36(11):2137-2143
With the discovery that transferrin serves as the iron source for hemoglobin-synthesizing immature red blood cells came the demonstration that a cell surface receptor, now known as transferrin receptor 1, is required for iron delivery from transferrin to cells. (A recently described second transferrin receptor, with as yet poorly understood function, will not be discussed in this brief review.) In succeeding years transferrin receptor 1 was established as a gatekeeper for regulating iron uptake by most cells, and the transferrin-to-cell endocytic pathway characterized in detail. HFE, the protein incriminated in the pathogenesis of hereditary hemochromatosis, a disorder of progressive and toxic iron overload, competes with transferrin for binding to receptor, thereby impeding the uptake of iron from transferrin. Mutation of HFE destroys this competition, thus facilitating access of transferrin and its iron to cells. Availability of the crystal structure of transferrin receptor 1, along with those of transferrin and HFE, opened research on molecular mapping of the transferrin-HFE- transferrin receptor interfaces by correlated synchrotron-generated hydroxyl radical footprinting and cryo-electron microscopy. The emerging challenge is to relate structure to the functional effects of receptor binding on the iron-binding and iron-releasing properties of transferrin within the iron-dependent cell. 相似文献
11.
Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron 总被引:6,自引:0,他引:6
A van der Ende A du Maine C F Simmons A L Schwartz G J Strous 《The Journal of biological chemistry》1987,262(18):8910-8916
Growing human choriocarcinoma BeWo b24 cells contain 1.5 X 10(6) functional cell surface transferrin binding sites and 2.0 X 10(6) intracellular binding sites. These cells rapidly accumulate iron at a rate of 360,000 iron atoms/min/cell. During iron uptake the transferrin and its receptor recycle at least each 19 min. The accumulated iron is released from the BeWo cells at a considerable rate. The time required to release 50% of previously accumulated iron into the extracellular medium is 30 h. This release process is cell line-specific as HeLa cells release very little if any iron. The release of iron by BeWo cells is stimulated by exogenous chelators such as apotransferrin, diethylenetriaminepenta-acetic acid, desferral, and apolactoferrin. The time required to release 50% of the previously accumulated iron into medium supplemented with chelator is 15 h. In the absence of added chelators iron is released as a low molecular weight complex, whereas in the presence of chelator the iron is found complexed to the chelator. Uptake of iron is inhibited by 250 microM primaquine or 2.5 microM monensin. However, the release of iron is not inhibited by these drugs. Intracellular iron is stored bound to ferritin. A model for the release of iron by BeWo cells and its implication for transplacental iron transport is discussed. 相似文献
12.
R Baynes G Bukofzer T Bothwell W Bezwoda B Macfarlane 《European journal of cell biology》1987,43(3):372-376
Transferrin receptors have been previously found on human macrophages and it has also been shown that transferrin iron is taken up by these cells. It has therefore been inferred that the uptake is receptor mediated and involves an endocytic pathway. The subject was addressed directly in the present study in which the transferrin-iron-receptor interaction was characterized in cultured human blood monocytes. Specific, saturable diferric transferrin binding was demonstrated, with a kDa of 3.6 X 10(-8) M and a calculated receptor density of 1.25-2.5 X 10(5) receptors per cell. Incubation at 4 degrees C markedly reduced transferrin binding and completely inhibited iron uptake. Chase experiments confirmed progressive cellular loading of iron, with concomitant loss of transferrin. Inhibitors of endocytic vesicle acidification (ammonium chloride and 2,4-dinitrophenol) inhibited iron unloading from endocytosed diferric transferrin, while microtubular inhibitors (colchicine and vindesine) and a microfilament inhibitor (cytochalasin B) reduced diferric transferrin uptake but had little effect on the iron unloading pathway. A similar effect was noted with a calcium ion antagonist (verapamil) and with 2 calmodulin antagonists (chlorpromazine and imipramine). These latter findings suggest the importance of cytoskeleton-membrane interactions via a calcium, calmodulin and protein kinase C mediated system. Endocytosed iron accumulated progressively as ferritin within the cultured monocytes. 相似文献
13.
Endocytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site 总被引:32,自引:0,他引:32
The transferrin receptor (TR) mediates cellular iron uptake by bringing about the endocytosis of transferrin. We investigated whether the cytoplasmic domain of 65 N-terminal amino acids or phosphorylated sites within this domain constitute a structure that is required for TR endocytosis. To test this hypothesis, we modified the cytoplasmic serine residues or introduced a deletion of 36 amino acids by in vitro mutagenesis of a cDNA expression vector for human TR. Upon expression in transfected mouse Ltk- cells, both the wild-type and phosphorylation site mutant receptors mediated transferrin internalization, whereas the truncated receptor did not. These results provide evidence that the cytoplasmic domain, or part of it, is essential for internalization of the TR, but argue against a role for receptor phosphorylation in endocytosis. 相似文献
14.
Transferrin receptor 2: a new molecule in iron metabolism 总被引:1,自引:0,他引:1
Transferrin receptor 1 (TfR1) which mediates uptake of transferrin-bound iron, is essential for life in mammals. Recently, a close homologue of human transferrin receptor 1 was cloned and called transferrin receptor 2 (TfR2). A similar molecule has been identified in the mouse. Human transferrin receptor 2 is 45% identical with transferrin receptor 1 in the extracellular domain, but contains no iron responsive element in its mRNA and is apparently not regulated by intracellular iron concentration nor by interaction with HFE. Transferrin receptor 2, like transferrin receptor 1, binds transferrin in a pH-dependent manner (but with 25 times lower affinity) and delivers iron to cells. However, transferrin receptor 2 distribution differs from transferrin receptor 1, increasing in differentiating hepatocytes and decreasing in differentiating erythroblasts. Expression of both receptors is cell cycle dependent. Mutations in the human transferrin receptor 2 gene cause iron overload disease, suggesting it has a role in iron homeostasis. 相似文献
15.
Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells. 总被引:5,自引:0,他引:5
Cindy N Roy Kenneth P Blemings Kathryn M Deck Paige S Davies Emily L Anderson Richard S Eisenstein Caroline A Enns 《Journal of cellular physiology》2002,190(2):218-226
Iron regulatory proteins (IRPs), the cytosolic proteins involved in the maintenance of cellular iron homeostasis, bind to stem loop structures found in the mRNA of key proteins involved iron uptake, storage, and metabolism and regulate the expression of these proteins in response to changes in cellular iron needs. We have shown previously that HFE-expressing fWTHFE/tTA HeLa cells have slightly increased transferrin receptor levels and dramatically reduced ferritin levels when compared to the same clonal cell line without HFE (Gross et al., 1998, J Biol Chem 273:22068-22074). While HFE does not alter transferrin receptor trafficking or non-transferrin mediated iron uptake, it does specifically reduce (55)Fe uptake from transferrin (Roy et al., 1999, J Biol Chem 274:9022-9028). In this report, we show that IRP RNA binding activity is increased by up to 5-fold in HFE-expressing cells through the activation of both IRP isoforms. Calcein measurements show a 45% decrease in the intracellular labile iron pool in HFE-expressing cells, which is in keeping with the IRP activation. These results all point to the direct effect of the interaction of HFE with transferrin receptor in lowering the intracellular labile iron pool and establishing a new set point for iron regulation within the cell. 相似文献
16.
Effect of aluminium on iron uptake and transferrin-receptor expression by human erythroleukaemia K562 cells. 总被引:1,自引:0,他引:1
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Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin. 相似文献
17.
R Gambari R Barbieri D Buzzoni F Bernardi G Marchetti F Amelotti R Piva L Viola L del Senno 《Biochimica et biophysica acta》1986,886(2):203-213
The receptor for transferrin plays an important role both in tumor cell growth and in hemoglobin synthesis. In this paper, we demonstrate that the monoclonal antibody 42/6 to human transferrin receptor inhibits iron uptake in the human leukemic K562 cell line and suppresses hemoglobin accumulation in K562 cells induced to erythroid differentiation by butyric acid. In contrast, only slight inhibitory effects were observed on cell proliferation of both uninduced and erythroid-induced K562 cells treated with the 42/6 monoclonal antibody. In addition, the 42/6 monoclonal antibody to human transferrin receptor does not inhibit butyric acid-induced accumulation of gamma-globin mRNA. The effect of the 42/6 monoclonal antibody on hemoglobin synthesis appears to be restricted to human cell lines, as murine Friend erythroleukemic cells undergo erythroid differentiation when cultured in the presence of hexamethylenebisacetamide plus the 42/6 monoclonal antibody. The findings reported in this paper suggest (a) a dissociation of iron transport and accumulation of heme molecules from the expression of globin genes and (b) a different requirement of iron uptake by different iron-dependent functions such as cell proliferation and hemoglobin expression. 相似文献
18.
Comparative studies of the binding and growth-supportive ability of mammalian transferrins in human cells 总被引:1,自引:0,他引:1
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. 相似文献
19.
Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells 总被引:3,自引:0,他引:3
Ikuta K Fujimoto Y Suzuki Y Tanaka K Saito H Ohhira M Sasaki K Kohgo Y 《Biochimica et biophysica acta》2000,1496(2-3):221-231
HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and beta(2)-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the (59)Fe uptake and release, and internalization and recycling of (125)I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron ((59)Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4. 3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5x10(5)/cell to 1. 2x10(5)/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with (125)I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron. 相似文献
20.
The mechanism(s) by which iron in blood is transported across the blood-brain barrier (BBB) remains controversial. Here we have examined the first step of this trans-cellular pathway, namely the mechanism(s) of iron uptake into human brain microvascular endothelial cells (hBMVEC). We show that hBMVEC actively reduce non-transferrin bound Fe(III) (NTBI) and transferrin-bound Fe(III) (TBI); this activity is associated with one or more ferrireductases. Efficient, exo-cytoplasmic ferri-reduction from TBI is dependent upon transferrin receptor (TfR), also. Blocking holo-Tf binding with an anti-TfR antibody significantly decreases the reduction of iron from transferrin by hBMVEC, suggesting that holo-Tf needs to bind to TfR in order for efficient reduction to occur. Ferri-reduction from TBI significantly decreases when hBMVEC are pre-treated with Pt(II), an inhibitor of cell surface reductase activity. Uptake of (59)Fe from (59)Fe-Tf by endothelial cells is inhibited by 50?% when ferrozine is added to solution; in contrast, no inhibition occurs when cells are alkalinized with NH(4)Cl. This indicates that the iron reduced from holo-transferrin at the plasma membrane accounts for at least 50?% of the iron uptake observed. hBMVEC-dependent reduction and uptake of NTBI utilizes a Pt(II)-insensitive reductase. Reductase-independent uptake of Fe(II) by hBMVEC is inhibited up to 50?% by Zn(II) and/or Mn(II) by a saturable process suggesting that redundant Fe(II) transporters exist in the hBMVEC plasma membrane. These results are the first to demonstrate multiple mechanism(s) of TBI and NTBI reduction and uptake by endothelial cells (EC) of the BBB. 相似文献