Biosynthesis of ferritin in rat hepatoma cells and rat livers. II. Binding of iron by ferritin protein.

Ferritin and its protein subunits in rat hepatoma cell clone M-5123-C1 were biosynthetically labeled with [14C]leucine and 59Fe. Radioimmunoassays of ferritin/apoferritin and of protein subunits in the free polyribosome, membrane-bound polyribosome, smooth membrane, and cytosol fractions were done with ferritin-specific and subunit-specific rabbit IgG antibodies at various time intervals after pulsing. Much more 59Fe was bound by ferritin/apoferritin than by subunits in all of the cell fractions. Binding of iron to subunits may have been a random process. When hepatoma cells were simultaneously pulse-labeled with 59Fe and [14C]leucine, uptake of much of the 59Fe by ferritin occurred relatively early, in comparison to incorporation of [14C]leucine, in all of the cell fractions examined. Thus, 59Fe was readily incorporated into pre-existing ferritin. We conclude that most, if not nearly all, of the iron is incorporated after assembly of protein subunits.     

Mucosal iron binding proteins and the inhibition of iron absorption by endotoxin

The uptake of iron from a tied off jejunal segment into the body after the injection of a 59Fe labeled test dose was decreased after the administration of endotoxin by about 80% in both normal and iron deficient animals.--In the iron deficient group the distribution of 59Fe in the cytosol fraction of jejunal mucosa between transferrin and ferritin was determined chromatographically; the amount of 59Fe in the ferritin fraction increased remarkably after the endotoxin treatment and the ratio of both was changed in favor of ferritin.--It is hypothesized that the association of the diversion of iron to the mucosal ferritin with the decrease of the transport of iron into the blood caused by endotoxin might be the consequence of abnormal oxidations in the mucosa measured by others in liver tissue.     

Amines as inhibitors of iron transport in rabbit reticulocytes

The effect of the known inhibitors of iron uptake, n-butylamine and NH4Cl, was examined at the molecular level to more precisely define the mechanisms by which these lysosomotropic agents block iron uptake by rabbit reticulocytes. Utilizing a rapid pulse-chase technique to follow the handling of a cohort of 59Fe, 125I-transferrin bound to rabbit reticulocytes, both amines were observed to have no effect on the cell-mediated release of 59Fe from internalized transferrin. The results indicated, however, that both agents acted to 1) retard the internalization of transferrin bound to transferrin receptors on the plasma membrane of reticulocytes, 2) retard the externalization of internalized transferrin, and 3) block the transport into the cytosol of iron released from transferrin.     

Mobilization of iron from specifically labeled reticulocyte ghosts.

Specifically labeled 59Fe ghosts have been prepared by incubation of whole reticulocytes with 59Fe3+-transferrin-CO3(2)-- followed by washing and ghost isolation. The binding of 59Fe by the membrane fraction is quite stable over a wide range of conditions, but iron mobilization occurs on incubation with chelating agents or cell lysate. The time course of 59Fe mobilization by unlabeled reticulocyte lysate exhibits five apparently zero-order phases. The rate of iron mobilization is linearly dependent on the concentration of 59Fe ghosts present in the incubation mixture. In contrast, the relative concentration of lysate appears to exhibit a saturation dependence with regard to membrane iron mobilization. Bathophenanthroline sulfonate follows a multiphasic time course of iron mobilization similar to that found with the lysate. Lysate from mature erythrocytes was found to mobilize iron with kinetics that are identical to reticulocyte lysate. The number and duration of the phases is independent of the mobilizing agent. The role of the membrane fraction in regulating the rate of iron release to cytosol was also investigated by the repetitive incubation of 59Fe ghosts with fresh lysate. The rate of 59Fe mobilization depended on the condition of the ghost with regard to prior 59Fe depletion. This publication emphasizes the active role of the membrane fraction in determining the rate at which iron will become available to the cytosol and the possibility that cytosol factors modulate the action of membrane bound components.     

Hepatic subcellular metabolism of heme from heme-hemopexin: incorporation of iron into ferritin

The subcellular distribution and metabolic fate of [⁵⁹Fe]heme-[¹²⁵I]-labeled hemopexin after receptor-mediated interaction with the liver was examined in the rat. After intravenous injection, [⁵⁹Fe]heme from the complex and ⁵⁹Fe from hepatic catabolism of this heme accumulate in the liver and undergo changes in their subcellular distribution over 2 hours. The amounts of [⁵⁹Fe]heme and particularly of ⁵⁹Fe increase in the cytosol while remaining constant or decreasing in membranous fractions. In contrast, [¹²⁵I]-labeled hemopexin associated with the liver during heme transport is always a small fraction of the dose and is not measurably catabolized under these conditions.Gel filtration of the cytosol showed that ⁵⁹Fe increased linearly with time in a high molecular weight fraction which was identified immunologically as ferritin. We conclude that heme transported by hemopexin is metabolized by the liver and the iron conserved.     

Ferrous iron uptake by rat duodenal brush border membrane vesicles: Effects of dietary iron level and competing minerals (Zn, Mn, and Ca)

We sought to confirm a recent report that Fe⁺² uptake into rat brush-border membrane vesicles is markedly increased by short-term consumption of iron-deficient diet, with no additional enhancement as the animal becomes functionally iron-deficient with continuing dietary Fe deprivation. In addition, we investigated whether previously observed in vivo absorption interactions of iron, zinc, and manganese occur in the brush border membrane vesicles uptake process, and whether short-term or long-term consumption of an iron-deficient diet affects the interaction at the uptake level. We did not observe any differences in Fe⁺² uptake between normal and iron-deficient brush border membrane vesicles, even when the iron status contrast was intensified by feeding a high iron versus iron-deficient diet for 3 weeks. Equimolar Zn⁺² and Mn⁺² decreased Fe⁺² uptake by 29 to 50% and 11 to 39%, respectively. Iron deficiency did not alter these effects. Equimolar Fe⁺² decreased Zn⁺² uptake by 13 to 22%. Calcium, included as a negative control, did not affect Fe⁺² uptake. Thus, some competition between Fe⁺² and similar divalent cations does occur at the level of the brush border membrane; the exact nature of this competition remains to be determined.     

Intestinal absorption of cobalt and iron: mode of interaction and subcellular distribution.

1. The absorption kinetic of 59Fe-(FeCl3) and 60CO-(CoCl2) 10 min after administration of increasing doses (0.5--1,000 nmoles metal) into tied-off duodenal segments of normal and iron-deficient rats shows saturation characteristic for both metals; in iron-deficient rats the absorption of both metals was enhanced. 2. The addition of increasing amounts of cobalt to the 59Fe-containing test solutions caused a decrease of the absorption of iron. 3. The study of the time dependence of this interaction in iron-deficient rats revealed, that cobalt inhibits the release of iron from mucosal cells into the blood, whereas the uptake of iron from the lumen into the mucosal cells did not differ from the controls without administration of cobalt. 4. The subcellular distribution of 59Fe and 60 Co in mucosal cell homogenates of iron-deficient rats after ultracentrifugation on a polyvinylpyrrolidone-CsCl solution shows a similar pattern for both metals; in the presence of cobalt the subcellular distribution of 59Fe is not changed. 5. From these results the conclusion is drawn that cobalt inhibits iron absorption not by an interference with iron binding sites on or in the luminal membranes of the mucosal cells but by an interaction with the releasing process at the contraluminal side.     

Preferential utilization in vitro of iron bound to diferric transferrin by rabbit reticulocytes.

59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.     

Characterization of siderophore-mediated iron transport inGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum is capable of utilizing iron from hydroxamate siderophores of different structural classes. The relative rates of iron transport for ferrichrome, ferrichrysin, ferrioxamine B, fusigen, ferrichrome A, rhodotorulic acid, coprogen B, dimerium acid and ferrirhodin were 100%, 98%, 74%, 59%, 49%, 35%, 24%, 12% and 11% respectively. Ferrichrome, ferrichrysine and ferrichrome A inhibited [⁵⁹Fe]ferrioxamine-B-mediated iron transport by 71%, 68% and 28% respectively when added at equimolar concentrations to the radioactive complex. The inhibitory mechanism of [⁵⁹Fe]ferrioxamine B uptake by ferrichrome was non-competitive (K _i 2.4 M), suggesting that the two siderophores do not share a common transport system. Uptake of [⁵⁹Fe]ferrichrome, [⁵⁹Fe]rhodotorulic acid and [⁵⁹Fe]fusigen was unaffected by competition with the other two siderophores or with ferrioxamine B. Thus,G. candidum may possess independent transport systems for siderophores of different structural classes. The uptake rates of [¹⁴C]ferrioxamine B and⁶⁷Ga-desferrioxamine B were 30% and 60% respectively, as compared to [⁵⁹Fe]ferrioxamine B. The specific ferrous chelates, dipyridyl and ferrozine at 6 mM, caused 65% and 35% inhibition of [⁵⁹Fe]ferrioxamine uptake. From these results we conclude that, although about 70% of the iron is apparently removed from the complex by reduction prior to being transported across the cellular membrane, a significant portion of the chelated ligand may enter the cell intact. The former and latter mechanisms seem not to be mutually exclusive.     

Acquisition of iron from transferrin regulates reticulocyte heme synthesis

Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59Fe from [59Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [59Fe]transferrin. Also, Fe-SIH stimulates [2-14C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59Fe incorporation into heme from either [59Fe]transferrin or [59Fe]SIH but does reverse the inhibition of 59Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes.     

Effect of orally-administered epidermal growth factor on intestinal iron absorption and mucosal permeability

A progressive increase in intestinal 59Fe3+ absorption was observed on oral feeding of mice with physiological doses of EGF/UGO. Maximal changes were apparent after 3d and appeared to be dose-dependent. In addition to a small increase in intestinal cell proliferation, as reflected by increased ornithine decarboxylase activity, EGF/UGO-feeding increased mucosal permeability (evaluated with [51Cr]-EDTA): the latter could account for the increase in iron absorption. Sialoadenectomy, to remove the major source of endogenous EGF/UGO, had no appreciable effect on the intestinal absorption of iron.     

Comparison of 59Fe3+ uptake in vitro and in vivo by mouse duodenum

Initial rates of 59Fe3+ uptake by mouse duodenal fragments (in vitro) and tied-off duodenal segments (in vivo) have been characterised for control and hypoxic animals. 59Fe3+ uptake by duodenal fragments was rapid, selective and dependent on medium Fe3+-nitrilotriacetate concentration. Most of the 59Fe3+ uptake (70-75%) occurred via the mucosal route and was dependent on the metabolic state of the tissue. Mucosal uptake showed an adaptive increase following exposure of animals to 3 days hypoxia; the enhancement was due to a 2-3-fold increase in Vmax app, without any significant changes in the Km app. Studies of upper small intestine transit times showed a mean residence time of 4-5 min for 59Fe-labelled mouse chow, emphasising the importance of initial uptake measurements. Time courses for in vivo total mucosal uptake exhibited linearity over a wide variety of absorption rates after correction for the permeation by intact metal-chelate complex. The corrected uptake showed a hyperbolic dependence on medium Fe3+-nitrilotriacetate concentration. Kinetic studies revealed a 2-3-fold increase in total mucosal uptake in hypoxia. Mucosa-to-carcass transfer of 59Fe was also markedly increased by chronic hypoxia. The in vitro system exhibits similar qualitative and quantitative kinetics for Fe3+ transport via the mucosal membrane to those obtained in vivo. The results observed in vitro are thus valid and provide a convenient method for further studies on Fe3+ transport in animals and in man.     

Role of plasma membrane phospholipids in the uptake and release of transferrin and its iron by reticulocytes

Summary The involvement of membrane phospholipids in the utilization of transferrinbound iron by reticulocytes was investigated using [⁵⁹Fe]- and [¹²⁵I]-labelled transferrin and rabbit reticulocytes which had been incubated with phospholipas A. Transferrin and iron uptake and release were all inhibited by phospholipas A which produced a marked decrease in the relative abundance of phosphatidylcholine and phosphatidylethanolamine and equivalent increases in their lyso-compounds in the reticulocyte plasma membrane. There was a close correlation between the iron uptake rate and the rate and amount of transferrin uptake and the amount of the lysophospholipids in the membrane. Incubation of the cells with exogenous lysophosphatidylethanolamine or lysophosphatidylcholine also produced inhibition of iron and transferrin uptake. The reduced uptake produced by phospholipase A could be reversed if the lyso-compounds were removed by fatty acid-free bovine serum albumin or by reincubation in medium 199. Treatment with phospholipase A was shown to increase the amount of transferrin bound by specific receptors on the reticulocyte membrane but to inhibit the entry of transferrin into the cells.The present investigation provides evidence that the phospholipid composition of the cell membrane influences the interaction of transferrin with its receptors, the processes of endocytosis and exocytosis whereby transferrin enters and leaves the cells, and the mechanism by which iron is mobilized between its binding to transferrin and incorporation into heme. In addition, the results indicate that phosphatidylethanolamine is present in the outer half of the lipid bilayer of reticulocyte membrane.     

Studies of Fe3+ transport across isolated intestinal brush-border membrane of the mouse

Mouse intestinal brush-border membrane vesicles take up iron from media containing 59Fe3 +-nitrilotriacetic acid. The iron uptake by the vesicles represents accumulation of iron which relates to an osmotically active space. Uptake is linearly related to vesicle protein concentration and is inhibited by low incubation temperature and low medium free Fe3+ concentrations. Experiments with the lipid soluble iron ligand 8-hydroxyquinoline and with Triton X-100 imply that the uptake is rate limited by membrane transport.     

Subcellular localization of recently-absorbed iron in mouse duodenal enterocytes: identification of a basolateral membrane iron-binding site

The subcellular distribution of newly absorbed iron in isolated mouse duodenal enterocytes was investigated by analytical subcellular fractionation using sucrose density gradient centifugation. Two major peaks of mucosal 59Fe activity were observed: one soluble and one particulate (density 1.18-1.20 g ml-1). The latter was increased following prior exposure of animals to chronic hypoxia. The particulate 59Fe was localized to the basolateral membranes using the marker enzyme Na+, K+ activated, Mg2+ dependent, ATPase and by washing intact enterocytes with the selective plasma membrane perturbant digitonin. The basolateral membrane can be selectively labelled by in vitro incubation of intact enterocytes at 0 degrees C with 59Fe(III)-nitrilotriacetate complex, confirming the presence of a 59Fe binding site on this membrane. No significant difference in in vitro iron binding to this site was observed between normal and chronically hypoxic animals. Iron binding to the basolateral membrane was significantly higher in disrupted, compared to intact enterocytes, indicating that this site is present on both sides of the basolateral membrane. It is therefore suggested that the increased labelling of this site in hypoxia, in vivo, is a consequence of an increase in a mucosal Fe pool which is available for binding to a membrane receptor.     

Membrane transport of non-transferrin-bound iron by reticulocytes

The transport of non-transferrin-bound iron into rabbit reticulocytes was investigated by incubating the cells in 0.27 M sucrose with iron labelled with 59Fe. In most experiments the iron was maintained in the reduced state, Fe(II), with mercaptoethanol. The iron was taken up by cytosolic, haem and stromal fractions of the cells in greater amounts than transferrin-iron. The uptake was saturable, with a Km value of approx. 0.2 microM and was competitively inhibited by Co2+, Mn2+, Ni2+ and Zn2+. It ceased when the reticulocytes matured into erythrocytes. The uptake was pH and temperature sensitive, the pH optimum being 6.5 and the activation energy for iron transport into the cytosol being approx. 80 kJ/mol. Ferric iron and Fe(II) prepared in the absence of reducing agents could also be transported into the cytosol. Sodium chloride inhibited Fe(II) uptake in a non-competitive manner. Similar degrees of inhibition was found with other salts, suggesting that this effect was due to the ionic strength of the solution. Iron chelators inhibited Fe(II) uptake by the reticulocytes, but varied in their ability to release 59Fe from the cells after it had been taken up. Several lines of evidence showed that the uptake of Fe(II) was not being mediated by transferrin. It is concluded that the reticulocyte can transport non-transferrin-bound iron into the cytosol by a carrier-mediated process and the question is raised whether the same carrier is utilized by transferrin-iron after its release from the protein.     

The subcellular distribution of iron in rabbit alveolar macrophages.

Rabbit alveolar macrophages were incubated with [⁵⁹Fe], washed, re-incubated with “cold” iron and homogenized. The distribution of radioactivity among the mitochondrial, lysosomal, microsomal and cytosol fractions was determined at short intervals after the onset of incubation. The findings indicate that the mitochondria form a significant iron-binding site during the early stage of iron uptake. A part of the mitochondrial-associated iron is later transferred to the cytosol where it is present in ferritin and in a low molecular weight form. Ferritin is the sole iron-binding protein of the cytosol.     

Microvillar iron-binding glycoproteins isolated from the rabbit small intestine.

Rabbit intestinal microvillus membranes possess high-affinity receptors for iron whose activity reflects homeostatic changes in mucosal iron transport. To isolate and characterize these membrane components, purified microvilli were radiolabelled with 59Fe(II) and solubilized in Triton X-100. 59Fe in 105000g supernatants co-eluted with a major broad protein peak (Mr approx. 100000) on gel-permeation chromatography and was rendered diffusible by Pronase digestion but not mild periodate degradation. Fluorescence studies with castor-bean lectin conjugates showed specific binding of this affinity probe exclusively to brush-border membranes in the intestinal epithelium. Affinity chromatography of solubilized membrane proteins showed binding to columns of immobilized lectin. Elution with D(+)-galactose released glycoprotein-bound 59Fe purified up to sevenfold over initial membrane extracts. The lectin bound up to 82% of protein-bound 59Fe. In contrast polyspecific antisera raised against rabbit microvilli in guinea-pigs precipitated less than 10% of solubilized radioactivity. Significantly more protein-bound 59Fe in detergent extracts of microvilli purified from bled animals interacted specifically with the lectin, suggesting that membrane glycoprotein receptors are involved in the homeostatic control of intestinal iron transport.     

Fe3+ transport by brush-border membrane vesicles isolated from normal and hypoxic mouse duodenum and ileum

Studies of 59Fe3+ uptake by brush-border membrane vesicles prepared from mouse duodenum have indicated that uptake represents transport across the brush-border membrane which is rate-limited by the membrane-transfer step (Simpson, R.J. and Peters, T.J. (1984) Biochim. Biophys. Acta 772, 220-226). Further studies presented here reveal that the uptake rate represents the net influx rate for Fe3+ and is independent of Na+ in the medium and of the method of vesicle preparation. Uptake by brush-border membrane vesicles prepared from mouse distal ileum also represents predominantly transport and is higher than that observed with duodenal brush-border membrane vesicles. Studies of the initial uptake rate by vesicles prepared from normal and hypoxic mouse intestine demonstrated an increase in Fe3+ transport in duodenal vesicles only.     

Role of membrane surface potential and other factors in the uptake of non-transferrin-bound iron by reticulocytes

Reticulocytes suspended in low ionic strength media such as isotonic sucrose solution efficiently take up non-transferrin-bound iron and utilize it for heme synthesis. The present study was undertaken to determine how such media facilitate iron utilization by the cells. The effects of changes in membrane surface potential, membrane permeability, cell size, transmembrane potential difference, oxidation state of the iron, and lipid peroxidation were investigated. Iron uptake to heme, cytosol, and stromal fractions of cells was measured using rabbit reticulo-cytes incubated with ⁵⁹Fe-labelled Fe(II) in 0.27 M sucrose, pH 6.5. Suspension of the cells in sucrose led to increased membrane permeability, loss of intracellular K⁺, decreased cell size, and increased transmembrane potential difference. However, none of these changes could account for the high efficiency of iron uptake which was observed. The large negative membrane surface potential which occurs in sucrose was modified by the addition of mono-, di-, tri-, and polyvalent cations to the solution. This inhibited iron uptake to a degree which for many cations varied with their valency. Other cations (Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺) were also very potent inhibitors, probably due to direct action on the transport process. Ferricyanide inhibited iron uptake, while ferrocyanide and ascorbate increased the uptake of Fe(III) but not Fe(II). It is concluded that the high negative surface potential of reticulocytes suspended in sucrose solution facilitates iron uptake by aiding the approach of iron to the transport site on the cell membrane. The iron is probably transported into the cell in the ferrous form. © 1994 wiley-Liss, Inc.