Reduced hepatic iron uptake from rat aglycotransferrin

Summary Rat aglycotransferrin (rAgTf) was produced from the disialosyl diantennary fraction of rat transferrin (rTf) by treatment with peptide:N-glycosidase F. Following removal of the enzyme by gel filtration and isolation of the deglycosylated protein by lectin chromatography, rAgTf was compared to rTf both in vitro and in vivo. No significant differences were found between the two proteins with respect to affinity for iron and kinetics of Fe release from the N-lobe and C-lobe. The fluorescence emission spectrum of apo-rTf was red-shfited by approximately 3 nm relative to diferric rTf; however, no spectral difference was detected between rTf and rAgTf when the analogous forms (apo or diferric) were compared. Plasma clearance of radioactive iron administered to rats as either rTf or rAgTf was comparable. Reticulocytes took up iron from rAgTf slightly faster than from rTf. In contrast, Fe acquisition by the liver from rAgTf was significantly reduced relative to rTf. This finding contrasts sharply with earlier observations with asialotransferrin (rAgTf) and provides a basis for discounting charge loss as the mechanism of enhanced hepatic Fe uptake from rAgTf. It is suggested that the glycan complement of rTf, while unimportant for interaction of the protein with specific receptors, probably plays a role in the interaction with low-affinity hepatic binding sites.     

Diacytosis of human asialotransferrin type 3 in the rat liver is due to the sequential engagement of two receptors

The possible role of transferrin receptors in the diacytosis of human asialotransferrin type 3 (HAsTf-3) by the rat liver was studied in vivo. A trace dose of the ligand was allowed to compete for hepatic binding sites against diferric transferrin, the concentration of which was varied between 5 400- and 18 000-fold. Binding of HAsTf-3 was insensitive to the presence of 2Fe-transferrin in this range, and the liver bound the ligand equally efficiently, regardless of whether it was presented in the holo or apo form. In contrast, pretreating the animals with desialylated bovine submaxillary mucin (2 mg/100 g, 2 min before the dose) prevented the asialotransferrin-liver interaction. These findings indicate that endocytosis of HAsTf-3 is mediated by the Gal/GalNAc-specific lectin and not by transferrin receptors. Although 2Fe-transferrin did not affect binding, it did reduce the half-life of the ligand in the liver, thus suggesting that transferrin receptors play an important role in the exocytic leg of the diacytic cycle. Based on our present and earlier data, a model is proposed in which the engagement of lectin and transferrin receptor in the diacytic cycle is envisaged sequentially so that HAsTf-3 switches receptors at an acidified subcellular site.     

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.     

Relationship between pinocytic rate and uptake of transferrin by suspended rat hepatocytes

Summary The aim of this study was to compare quantitatively the capacity to transcytose (i.e. to uptake and release) transferrin (Tf) with the pinocytic activity of suspended adult rat hepatocytes. An oligodisperse preparation of¹³¹I-polyvinylpyrrolidone (PVP;M r 36000) was used to measure the inward and outward aspects of the pinocytic process in separate experiments. Cell association of rat¹²⁵I-Tf was measured at Tf concentrations approaching physiological, where⁵⁹Fe uptake obeyed first-order kinetics. Release studies with both PVP and Tf were carried out under conditions which minimized the probability ofde novo endocytosis of a molecule already released. Sets of experimental points representing cell-associated radioactivities were converted into continuous algebraic functions by fitting with two-term (release studies) or three-term (uptake studies) exponential equations. Transport of PVP and Tf through the cells was computed from these equations by deconvolution. This analysis showed that, under the present experimental conditions, the fractional transcytosis rates of Tf and PVP by hepatocytes were in the ratio of I:0.77. These values imply that, in the physiological range of Tf concentrations, about 75% of the Fe taken up by hepatocytes may be due to a pinocytic mechanism (fluid-phase or mixed). Inclusion of chloroquine (1 mM) in the suspending medium, both in uptake and release experiments, resulted in more PVP and Tf passing through the cells, while Fe uptake was reduced. It is suggested that the base probably exerted its enhancing effect on transcytosis by shunting the subcellular transport of PVP and Tf to the outward leg through a shorter circuit.Abbreviations BSA bovine serum albumin - HBSS Hank's balanced salt solution - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MEM minimal essential medium - PVP polyvinylpyrrolidone - Tf transferrin     

Differences in transferrin response and numbers of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potentials

We previously found that transferrin (Tf) differentially stimulated the growth of highly metastatic variant lines of murine melanoma and that these highly metastatic cells also had greater numbers of Tf receptors on their cell surfaces. In the present study we found that highly metastatic rat mammary adenocarcinoma cell lines also responded differentially to Tf in proliferation assays, and cell monolayers bound Tf in relation to their metastatic potential (MTPaB10 > MTPaB5 > MTLn3 > MTLn2 > MTC > MTF7 > MTPa). The brain-colonizing lines PaB10 and PaB5 were the most responsive to Tf and had the highest numbers of Tf receptors. Different human breast cancer cell lines also responded differentially to Tf in proliferation assays and bound different amounts of Tf to their cell surface Tf receptors. Transferrin binding, but not growth response, correlated with metastatic and invasive properties of lines selected from the human MCF-7 series (MCF7/LCC2 > MCF7/LCC1 > MCF7). In examining the transferrin binding and growth response of lines from the human MDA series, the Tf binding and growth response was MDA231 > MDA435 > MDA468. The lines MDA435 and MDA231 were metastatic in nude mouse assays, whereas the line MDA468 was not. Scatchard analysis indicated the presence of a single class of receptor for Tf on the rat and human mammary cell lines. The results suggest that neoplastic cells displaying various metastatic properties may express differing numbers of Tf receptors and respond differently to growth factors such as Tf. © 1993 Wiley-Liss, Inc.     

Rat aglycotransferrin and human monoglycotransferrin: production and metabolic properties

Rat transferrin (rTf), containing one complex glycan, and human transferrin (hTf), containing two complex glycans, were treated with peptide:N-glycosidase F (PNGase) under nondenaturing conditions. Apo rTf with a nonfucosylated standard biantennary glycan, but not its diferric counterpart, yielded satisfactory amounts (approximately 55% in 7 h) of aglyco Tf (AgTf). The presence of a chitobiose core fucose reduced yields to approximately 30%, whereas an additional NeuAc on the GlcNAc in the Man(alpha 1-3) branch had no adverse effect. Triton X-100 impaired deglycosylation. The main product (approximately 65%) obtained from apo hTf was monoglyco Tf (MgTf). Analysis of the cyanogen bromide fragments of MgTf revealed that PNGase did not discriminate between the two glycosylation sites of hTf. A negligible portion (2-4%) of AgTf, that was also obtained during the reaction, probably resulted from PNGase action on denatured hTf molecules. Modified Tfs were separated by affinity chromatography, radiolabeled, mixed with another preparation of interest, and injected in rats. Total-body radiation measurements showed that the half-life of rat AgTf was 19-20% shorter than that of rTf but 9% longer than that of asialo Tf. This suggests that close to 76% of the change in the degradative rate observed after desialylating rTf is referable to charge loss rather than the exposure of Gal residues. Human MgTf was catabolized by rats 8-9% faster than the parent protein, while human AgTf behaved in vivo like a denatured protein. It is concluded that sialyl carboxyls are a codeterminant of the normal lifetime of transferrins.     

Transferrin and iron uptake by rat reticulocytes

The uptake of transferrin labeled with 3H and 59Fe by rat reticulocytes was studied to clarify the characteristics of the uptake process and intracellular transport. Rat reticulocytes took up transferrin in a saturable, time- and temperature-dependent manner. Scatchard analysis of the binding parameters indicated that transferrin molecules were bound to cell-surface receptors with high affinity. Monodansyl- cadaverine, a potent inhibitor of transglutaminase, reduced the amount of internalized transferrin but has no effect on the total amount of cell-associated transferrin, suggesting that transferrin is taken up by rat reticulocytes via receptor-mediated endocytosis. About 50% of the internalized 3H label was released from the cells after reincubation for 1 h in fresh medium. In contrast, no release of 59Fe label was observed. By immunoprecipitation and subsequent SDS-PAGE the released 3H-labeled product was identified as apotransferrin. Lysosomotropic reagents and a proton ionophore reduced the uptake of 59Fe. These results indicated that iron was removed from transferrin at an intracellular site in an acidic environment. The released iron was found not to associate with any intermediate ligands before it was utilized for heme synthesis in mitochondria.     

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

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

Specificity of hepatic iron uptake from plasma transferrin in the rat.

1. The role of specific interaction between transferrin and its receptors in iron uptake by the liver in vivo was investigated using 59Fe-125I-labelled transferrins from several animal species, and adult and 15-day rats. Transferrin-free hepatic uptake of 59Fe was measured 2 or 0.5 hr after intravenous injection of the transferrins. 2. Rat, rabbit and human transferrins gave high and approximately equal levels of hepatic iron uptake while transferrins from a marsupial (Sentonix brachyurus), lizard, crocodile, toad and fish gave very low uptake values. Chicken ovotransferrin resulted in higher uptake than with any other species of transferrin. 3. Iron uptake by the femurs (as a sample of bone marrow erythroid tissue) and, in another group of 19-day pregnant animals by the placentas and fetuses, was also measured, for comparison with the liver results. The pattern of uptake from the different transferrins was found to be similar to that of iron uptake by the liver except that with femurs, placentas and fetuses ovotransferrin gave low values comparable to those of the other non-mammalian species. 4. It is concluded that iron uptake by the liver from plasma transferrin in vivo is largely or completely dependent on specific transferrin-receptor interaction. The high hepatic uptake of iron from ovotransferrin was probably mediated by the asialoglycoprotein receptors on hepatocytes.     

Transferrin-bound and transferrin free iron uptake by cultured rat astrocytes.

Previously we had demonstrated the presence of transferrin receptor (TfR) on the plasma membrane of cultured rat cortical astrocytes. In this study, we investigated the roles of TfR in transferrin-bound iron (Tf-Fe) as well as transferrin-free iron (Fe II) uptake by the cells. The cultured rat astrocytes were incubated with 1 microM of double-labelled transferrin (125I-Tf-59Fe) in serum- free DMEM F12 medium or 59Fe II in isotonic sucrose solution at 37 degrees C or 4 degrees C for varying times. The cellular Tf-Fe, Tf and Fe II uptake was analyzed by measuring the intracellular radioactivity with gamma counter. The result showed that Tf-Fe uptake kept increasing in a linear manner at least in the first 30-min. In contrast to Tf-Fe uptake, the internalization of Tf into the cells was rapid initially but then slowed to a plateau level after 10 min. of incubation. The addition of either NH4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release from Tf within endosomes, decreased the cellular Tf-Fe uptake but had no significant effect on Tf uptake. Pre-treated cells with trypsin inhibited significantly the cellular uptake of Tf-Fe as well as Tf. These findings suggested that Tf-Fe transport across the membrane of astrocytes is mediated by Tf-TfR endocytosis. The results of transferrin-free iron uptake indicated that the cultured rat cortical astrocytes had the capacity to acquire Fe II. The highest uptake of Fe II occurred at pH 6.5. The Fe II uptake was time and temperature dependent, iron concentration saturable, inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+ and not significantly affected by phenylarsine oxide treatment. These characteristics of Fe II uptake by the cultured astrocytes suggested that Fe II uptake is not mediated by TfR and implied that a carrier-mediated iron transport system might be present on the membrane of the cultured cells.     

Effects of sialic acid residues of transferrin on the binding with aluminum and iron studied by HPLC/high-resolution ICP-MS

Transferrins (Tfs) are glycoproteins with carbohydrate chains in the C-lobe. Carbohydrate-deficient Tfs (CDTs) with fewer sialic acids increased in several diseases. In this study, the affinity of metals (Al and Fe) to Tfs was compared between native- and asialo-Tf by on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry, to clarify whether the presence of sialic acids influences the metal binding. Fe added as Fe-citrate in the presence of bicarbonate preferred the N-lobe site and the binding affinity was similar between native- and asialo-Tfs. Al-citrate added at Al/Tf = 1 also preferred the N-lobe site, while the binding affinity was higher to asialo-Tf than to native-Tf. In Al-oxalate addition, the affinity to the N-lobe site of both Tfs increased further. In the absence of bicarbonate, Al-oxalate showed a preference for the C-lobe site in native-Tf and comparable affinity to both lobes in asialo-Tf. In asialo-Tf, Al2-Tf was the largest peak even at Al/Tf = 1. Thus, the lack of sialic acid in glycans and the presence of oxalate enhanced the binding affinity of Al to Tf. Therefore, it was suggested that the binding affinity of Al in patients with CDTs may be enhanced.     

An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium

Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway--receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu(C)Fe(N)Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.     

Role of transferrin in iron uptake by the brain: a comparative study

Summary The role of specific transferrin (Tf) and Tf receptor interaction on brain capillary endothelial cells in iron transport from the plasma to the brain was investigated by using Tf from several species of animals labeled with ⁵⁹Fe and ¹²⁵I, and 15-day and adult rats. The rate of iron transfer was much greater in the 15-day rats. It was greatest with Tf from the mammals, rat, rabbit and human, but much lower with chicken ovotransferrin and quokka (a marsupial), toad, lizard, crocodile, and fish Tf. The uptake of Tf by the brain showed a similar pattern, except for a very high uptake of ovotransferrin (ovo Tf). Iron uptake by the femurs (a source of bone marrow) was also high with Tf from the mammalian species and low with the other types of Tf, but showed little change with aging of the animals. It is concluded that iron transport into the brain is dependent on the function of Tf receptors, probably on capillary endothelial cells, and that these receptors show the same type of species specificity as the receptors on immature erythroid cells. Also, the decrease in iron uptake by the brain as rats age from 15 days to adulthood is specific for the brain and is not a general effect of the aging process.Abbreviations Tf transferrin - ovo Tf ovotransferrin     

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.     

Uptake of sialo and asialo transferrins by isolated rat hepatocytes. Comparison of a heterologous and a homologous system

Incubation of isolated rat hepatocytes with different human sialo transferrins shows that interaction with the specific transferrin receptor is insensitive to differences in the carbohydrate composition of the glycans. Asialo transferrins lead to an increased iron uptake, which is dependent on the amount of exposed galactose. This is explained by the presence of the asialo glycoprotein (AsGP) receptor. Experiments with selective saturation of the two receptor systems show that on incubation with human asialo transferrin (AsHTf) transferrin uptake proceeds increasingly via the AsGP receptor on raising the concentration. Homologous rat asialo transferrin (AsRTf) behaves similarly, but less pronounced. Iron is accumulated via both receptor systems in the heterologous system, but only via the transferrin receptors in the homologous system. The difference in interaction with the AsGP-receptor may be caused by the difference in galactose content of the two asialo transferrins. As an explanation for the differences in intracellular metabolism a hitherto unknown recognition system for species specificity is postulated which protects homologous AsRTf from degradation, but directs foreign AsTf to lysosomes.     

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.     

Uptake and subcellular processing of 59Fe-125I-labelled transferrin by rat liver.

The uptake of transferrin and iron by the rat liver was studied after intravenous injection or perfusion in vitro with diferric rat transferrin labelled with 125I and 59Fe. It was shown by subcellular fractionation on sucrose density gradients that 125I-transferrin was predominantly associated with a low-density membrane fraction, of similar density to the Golgi-membrane marker galactosyltransferase. Electron-microscope autoradiography demonstrated that most of the 125I-transferrin was located in hepatocytes. The 59Fe had a bimodal distribution, with a larger peak at a similar low density to that of labelled transferrin and a smaller peak at higher density coincident with the mitochondrial enzyme succinate dehydrogenase. Approx. 50% of the 59Fe in the low-density peak was precipitated with anti-(rat ferritin) serum. Uptake of transferrin into the low-density fraction was rapid, reaching a maximal level after 5-10 min. When livers were perfused with various concentrations of transferrin the total uptakes of both iron and transferrin and incorporation into their subcellular fractions were curvilinear, increasing with transferrin concentrations up to at least 10 microM. Analysis of the transferrin-uptake data indicated the presence of specific transferrin receptors with an association constant of approx. 5 X 10(6) M-1, with some non-specific binding. Neither rat nor bovine serum albumin was taken up into the low-density fractions of the liver. Chase experiments with the perfused liver showed that most of the 125I-transferrin was rapidly released from the liver, predominantly in an undegraded form, as indicated by precipitation with trichloroacetic acid. Approx. 40% of the 59Fe was also released. It is concluded that the uptake of transferrin-bound iron by the liver of the rat results from endocytosis by hepatocytes of the iron-transferrin complex into low-density vesicles followed by release of iron from the transferrin and recycling of the transferrin to the extracellular medium. The iron is rapidly incorporated into mitochondria and cytosolic ferritin.     

Distinction between binding and endocytosis of human asialo-transferrin by the rat liver

The ability of the rat liver to bind and endocytose human asialo-transferrin was investigated in vivo. Asialo-transferrin was separated from incompletely desialylated transferrin and neuraminidase by chromatography before being labelled with ¹²⁵I. Plasma radioactivity curves and hepatic radioactivity contents measured over a 1270-fold dose range led to the following observation. At the lowest dose (0.4μg/100g body wt.), the distribution of asialo-transferrin between plasma and liver resembled a reversible reaction reaching equilibrium in approx. 20min. After 35min, 93% of the dose was recovered with the plasma and liver as protein-bound radioactivity. Most of the asialo-transferrin associated with the liver could be displaced by asialo-orosomucoid, indicating that binding of asialo-transferrin to the galactose-specific lectin on the plasma membrane of hepatocytes was not followed by a signal for endocytosis. A range of doses, up to an average of 509.2μg of asialo-transferrin per 100g body wt., resulted in progressive increments in asialo-transferrin catabolism, as evidenced by lower dose recoveries and increased concentrations of non-protein-associated radioactivity in the liver and plasma volume. These observations indicate that binding and endocytosis of human asialo-transferrin by the rat hepatocyte are distinct phenomena. Individual asialo-transferrin molecules, although readily bound by the hepatic lectin, lack either the quantity or spacing of terminal galactose residues necessary for triggering endocytosis. Although endocytosis is induced by several asialo-transferrin molecules acting synergistically, preliminary experiments with asialo-glycopeptides and other substances have so far failed to provide further insight into the chemical basis of the signal for endocytosis.     

Transferrin receptors of isolated rat seminiferous tubules bind both rat and human transferrin

The binding and uptake of rat and human transferrin by isolated rat seminiferous tubules was studied. During the isolation and incubation of the tubules, the blood-testis barrier remained intact. Iron-saturated and iron-free (apo-) transferrin use the same binding sites on the surface of the tubules, but the dissociation constant is about two times higher for apotransferrin than for iron-saturated transferrin. The affinity of the receptors is equal for rat and human transferrin, but human transferrin binds to more surface binding sites (2.6 X 10(10) per 10 cm tubule length) than rat transferrin (1.1 X 10(10) per 10 cm tubule length) at 0 degrees C. At 33 degrees C equal numbers of human and rat transferrin molecules are taken up (about 8 X 10(10)) per 10 cm tubule length. The quantitative difference between 0 degrees C and 33 degrees C is caused by the fact that at 33 degrees C receptor-mediated endocytosis and recycling occur. As a consequence, both surface and intracellular transferrin receptors are detected at 33 degrees C. The dissociation constants are not temperature-dependent.     

Detection and isolation of a hepatic membrane receptor for ferritin

A ferritin receptor has been detected on isolated rat hepatocytes and has been partially purified from rat liver using affinity chromatography. Isolated hepatocytes exhibit approximately 30,000 ferritin binding sites/cell with a binding association constant (Ka) of 1 x 10(8) mol-1 liter. A binding assay has been developed which utilizes a hepatic ferritin receptor coupled to a microparticulate support to facilitate separation of bound and free ligand. This method yielded a Ka of 3 x 10(8) mol-1 liter for the purified hepatic ferritin receptor. Binding of ferritin to the insolubilized receptor was partially inhibited by human lactoferrin but unaffected by 200-fold molar excess of bovine albumin, rat transferrin, or human asialoorosomucoid.