Ferrous iron uptake byBifidobacterium breve

Bifidobacterium breve transports ferrous iron in preference to the ferric form in a saturable, concentration-dependent manner with an optimum pH of 6. Iron transport is highly temperature sensitive. Two transport systems with apparent Km's of 86 +/- 27 and 35 +/- 20 microM (p greater than 0.01) were distinguished, one operating at high iron concentrations, the other at low iron concentrations. Iron uptake could not be accounted for by surface binding. Uptake of iron was inhibited by iron chelators, a protein ionophore, and ATPase inhibitors, and it was stimulated by potassium ionophores. The presence of a ferri reductase in the insoluble cell fraction of B. breve and its "spent" growth medium was demonstrated. The hypothesis is presented that iron uptake by bifidobacteria is related to the nutritional immunity phenomenon.     

Transferrin iron interactions with cultured hepatocellular carcinoma cells (PLC/PRF/5)

Hepatocellular carcinoma cells of the PLC/PRF/5 cell line had 1.9 x 10(5) transferrin receptors per tumor cell with a Kd of 1.5 x 10(-8) M. At high concentrations of transferrin the binding was not saturable. Transferrin internalization by hepatoma cells was shown by time and temperature-dependent binding studies and by pronase experiments. Transferrin recycling was confirmed by the demonstration of a progressive increase in the cellular molar ratios of iron to transferrin and by chase experiments. Ammonium chloride interfered with iron unloading. The vinca alkaloid vincristine inhibited iron and transferrin uptake. The hepatocarcinoma cells appeared to lack asialoglycoprotein receptors and therefore internalized partially desialated transferrin by the regular route. Iron uptake from transferrin was markedly inhibited by the hydrophobic ferrous chelator 2,2' bipyridine but was relatively unaffected by the hydrophilic ferric chelator desferroxamine. The implication that ferrous iron was involved in postendocytic transvesicular membrane iron transport was supported by a study in which hepatoma cells were shown to take up large amounts of ferrous iron suspended in 270 mM sucrose at pH 5.5. The interaction at this pH between surface labeled hepatoma cell extracts and ferrous iron on a Sephacryl S-300 column suggested that the postendocytic transvesicular transport of iron through the membrane was in part protein mediated. The endocytosed iron in hepatoma cells was found in association with ferritin (33%), transferrin (31%) and a low molecular weight fraction (21%).     

Iron uptake mechanism in the chrysophyte microalga Dinobryon

The mechanism of iron uptake in the chrysophyte microalga Dinobryon was studied. Previous studies have shown that iron is the dominant limiting elements for growth of Dinobryon in the Eshkol reservoir in northern Israel, which control its burst of bloom. It is demonstrated that Dinobryon has a light-stimulated ferrireductase activity, which is sensitive to the photosynthetic electron transport inhibitor DCMU and to the uncoupler CCCP. Iron uptake is also light-dependent, is inhibited by DCMU and by CCCP and also by the ferrous iron chelator BPDS. These results suggest that ferric iron reduction by ferrireductase is involved in iron uptake in Dinobryon and that photosynthesis provides the major reducing power to energize iron acquisition. Iron deprivation does not enhance but rather inhibits iron uptake contrary to observations in other algae.     

Iron uptake in reticulocytes. Inhibition mediated by the ionophores monensin and nigerisin

The lipophilic carboxylic ionophores monensin and nigerisin reversibly blocked iron uptake by erythroid cells. At low concentrations of ionophores (0.25-0.5 microM), the disruption of the compartment in which iron is released affected minimally the release of iron from transferrin but effectively inhibited iron uptake. Iron released from transferrin was extruded from the cell synchronously with but not bound to transferrin. The compartment disrupted by the ionophores, and in which iron is released from transferrin, is apparently contiguous to the extracellular medium. Contiguity was assessed by determining the effect of extracellular Na+ and K+ on the activity of the ionophores. The above data fit a model of iron uptake in which iron is released from transferrin in an acidic compartment in immediate contiguity with the cell plasma membrane. Iron is then bound by its membrane acceptor and is translocated to the cytosolic side of the plasma membrane. At submicromolar concentrations, the ionophores monensin and nigerisin produce a small increase in the pH of the acidic compartment. The pH change, which is not sufficient to block the release of iron from transferrin, is enough to block the binding of released iron to its acceptor in the plasma membrane, thus producing inhibition of iron uptake.     

Involvement of cytochrome a in iron oxidation of a moderately thermophilic iron-oxidizing bacterium, strain TI-1.

The iron-oxidizing activity of a moderately thermophilic iron-oxidizing bacterium, strain TI-1, was located in the plasma membrane. When the strain was grown in Fe2+ (60 mM)-salts medium containing yeast extract (0.03%), the plasma membrane had iron-oxidizing activity of 0.129 mumol O2 uptake/mg/min. Iron oxidase was solubilized from the plasma membrane with 1.0% n-octyl-beta-D-glucopyranoside (OGL) containing 25% (v/v) glycerol (pH 3.0) and purified 37-fold by a SP Sepharose FF column chromatography. Iron oxidase solubilized from the plasma membrane was stable at pH 3.0, but quite unstable in the buffer with the pH above 6.0 or below 1.0. The optimum pH and temperature for iron oxidation were 3.0 and 55 degrees C, respectively. Solubilized enzyme from the membrane showed absorption peaks characteristic of cytochromes a and b. Cyanide and azide, inhibitors of cytochrome c oxidase, completely inhibited iron-oxidizing activity at 100 microM, but antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and myxothiazol, inhibitors of electron transport systems involved with cytochrome b, did not inhibit enzyme activity at 10 microM. The absorption spectrum of the most active enzyme fraction from SP Sepharose FF column chromatography (4.76 mumol O2 uptake/mg/min) compared with lower active fractions from the chromatography (0.009 and 2.10 mumol O2 uptake/mg/min) showed a large alpha-peak of cytochrome a at 602 nm and a smaller alpha-peak of cytochrome b at 560 nm. The absorption spectrum of pyridine ferrohemochrome prepared from the most highly purified enzyme showed an alpha-peak characteristic of heme a at 587 nm, but not the alpha-peak characteristic of heme c at 550 nm. The cytochrome a, but not cytochrome b, in the most highly purified enzyme fraction was reduced by the addition of ferrous iron at pH 3.0, indicating that electrons from Fe2+ were transported to cytochrome a, but not cytochrome b. These results strongly suggest that cytochrome a, but not cytochromes b and c, is involved in iron oxidation of strain TI-1.     

Mechanisms of ferric and ferrous iron uptake by Bifidobacterium bifidum var. pennsylvanicus

Iron uptake studies in Bifidobacterium bifidum var. pennsylvanicus were carried out using ferric citrate at iron concentrations above 0.01 mM and pH 7, ferrous iron at concentrations less than 0.01 mM at pH 5. Two ferric iron transport systems were distinguished: the temperature-insensitive polymer, and the temperature-sensitive monomer uptake. Both showed a saturation phenomenon. The transport of ferrous iron at concentrations below 0.01 mM was temperature-dependent, and its affinity for iron was higher than that of a system operating at iron concentrations higher than 0.01 mM. The use of various metabolic inhibitors indicated that ferrous iron transport at pH 5 at both high and low iron concentrations was mediated by transport-type ATPase. Proton gradient dissipators abolished ferrous iron uptakes as well as the ferric monomer uptake. Uptake of the ferric polymer was insensitive to metabolic inhibitors. The functional significance of the various types of iron transport systems may be related to the nutritional immunity phenomenon.     

Iron transport mechanisms in reticulocytes and mature erythrocytes

The mechanism of iron transport into erythroid cells was investigated using rabbit reticulocytes and mature erythrocytes incubated with ⁵⁹Fe-labelled Fe(II) in isotonic sucrose or in solutions in which the sucrose was replaced with varying amounts of isotonic NaCl or KCl. Iron uptake was inhibited at all concentrations of NaCl, in a concentration-dependent manner, but with KCl inhibition occurred only at concentrations up to 10 mM. Higher KCl concentrations stimulated iron uptake to the cytosol of the cells, but inhibited its incorporation into heme. This effect became more marked as the iron concentration was raised. It was found that KCl inhibits iron incorporation into heme and stimulates iron uptake by mature erythrocytes, as well as by reticulocytes. It is concluded that erythroid cells can take up nontransferrin-bound Fe(II) by two mechanisms. One is a high-affinity mechanism that is limited to reticulocytes, saturates at a low iron concentration, and is inhibited by metabolic inhibitors. The other is a low-affinity process that is found in both reticulocytes and erythrocytes, becomes more prominent at higher iron concentrations, and is stimulated by KCl, as well as RbCl, LiCl, CsCl, and choline Cl. The KCl stimulation is inhibited by amiloride, but not by metabolic inhibitors, and its operation is not dependent on changes in cell volume or membrane potential, but it does require the presence of a permeant extracellular anion. Iron uptake by this process appears to occur by facilitated transport and is possibly assoicated with exchange of Na⁺. A further aspect of this study was a comparison of iron uptake by reticulocytes from Fe(II)-sucrose and Fe(II)-ascorbate using a variety of incubation conditions. No major differences were observed. © 1995 Wiley-Liss, Inc.     

The mechanism of ferrous iron binding by suspensions of Bifidobacterium bifidum var. pennsylvanicus

Bifidobacterium bifidum var. pennsylvanicus acquires ferrous iron at a reasonable rate when this bacterium — in the resting state — is incubated with ferrous iron in either a modified Hanks' solution, ot Norris medium in which the bacteria had previously grown. Ferrous iron binding was substantially lower in unused Norris medium containing human milk whey, because it contained iron binding inhibitors that were degraded during bacterial growth. The inhibitors were identified as human milk whey components and bovine casein digest. The affinity of bacteria for ferrous iron was dependent on the iron concentration of the media. In modified Hanks' solution, the affinity of cells for iron at low iron concentrations was some 250-fold greater than that at high iron concentrations. Ferrous iron binding did not take place it a carbon source was omitted from the media or if the cells had previously been heated at 80°C for 20 min. Surface binding of ferrous iron to the bacterial membrane was minimal. Trypsin and microbial proteinase treatment of bacteria did not inhibit iron uptake, making an iron-specific receptor on bifidobacterial cell membranes unlikely. Iron binding by bifidobacteria is an energy-requiring process dependent upon the operation of a heat-sensitive enzyme machinery, and it does not involve an extracellular iron carrier.     

The effects of pH and the iron redox state on iron uptake in the intestine of a marine teleost fish, gulf toadfish (Opsanus beta)

In the marine teleost intestine the secretion of bicarbonate increases pH of the lumen (pH 8.4 -9.0) and importantly reduces Ca2+ and Mg2+ concentrations by the formation of insoluble divalent ion carbonates. The alkaline intestinal environment could potentially also cause essential metal carbonate formation reducing bioavailability. Iron accumulation was assessed in the Gulf toadfish (Opsanus beta) gut by mounting intestine segments in modified Ussing chambers fitted to a pH-stat titration system. This system titrates to maintain lumen pH constant and in the process prevents bicarbonate accumulation. The luminal saline pH was clamped to pH 5.5 or 7.0 to investigate the effect of proton concentrations on iron uptake. In addition, redox state was altered (gassing with N2, addition of dithiothreitol (DTT) and ascorbate) to evaluate Fe3+ versus Fe2+ uptake, enabling us to compare a marine teleost intestine model for iron uptake to the mammalian system for non-haem bound iron uptake that occurs via a ferrous/proton (Fe2+/H+) symporter called Divalent Metal Transporter 1 (DMT1). None of the redox altering strategies affected iron (Fe3+ or Fe2+) binding to mucus, but the addition of ascorbate resulted in a 4.6-fold increase in epithelium iron accumulation. This indicates that mucus iron binding is irrespective of valency and suggests that ferrous iron is preferentially transported across the apical surface. Altering luminal saline pH from 7.0 to 5.5 did not affect ferric or ferrous iron uptake, suggesting that if iron is entering via DMT1 in marine fish intestine this transporter works efficiently under circumneutral conditions.     

Transferrin as a donor of iron to mitochondria. Effect of pyrophosphate and relationship to mitochondrial metabolism and heme synthesis

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The uptake has a very low energy dependence, but it is highly dependent on a functioning respiratory chain. Reduction of the ferric-iron-pyrophosphate complex is not linked to any specific respiratory complex. Half of the amount of iron accumulated is passed into heme. Iron once accumulated is very little accessible to chelation by added ferric or ferrous iron chelators. Iron uptake and heme synthesis are maximal if a suitable porphyrin substrate is added simultaneously with iron. The results represent further evidence that pyrophosphate is a possible candidate for intracellular iron transport. Also, the results suggest that iron uptake is coupled to simultaneous porphyrin uptake and heme synthesis.     

Transferrin as a donor of iron to mitochondria Effect of pyrophosphate and relationship to mitochondrial metabolism and heme synthesis

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The uptake has a very low energy dependence, but it is highly dependent on a functioning respiratory chain. Reduction of the ferric-iron-pyrophosphate complex is not linked to any specific respiratory complex. Half of the amount of iron accumulated is passed into heme. Iron once accumulated is very little accessible to chelation by added ferric or ferrous iron chelators. Iron uptake and heme synthesis are maximal if a suitable porphyrin substrate is added simultaneously with iron. The results represent further evidence that pyrophosphate is a possible candidate for intracellular iron transport. Also, the results suggest that iron uptake is coupled to simultaneous porphyrin uptake and heme synthesis.     

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.     

Uptake of iron from transferrin by isolated rat hepatocytes. A redox-mediated plasma membrane process?

The uptake of iron from transferrin by isolated rat hepatocytes varies in parallel with plasma membrane NADH:ferricyanide oxidoreductase activity, is inhibited by ferricyanide, ferric, and ferrous iron chelators, divalent transition metal cations, and depends on calcium ions. Iron uptake does not depend on endosomal acidification or endocytosis of transferrin. The results are compatible with a model in which iron, at transferrin concentrations above that needed to saturate the transferrin receptor, is taken up from transferrin predominantly by mechanisms located to or contiguous with the plasma membrane. The process involves labilization and reduction of transferrin-bound iron by cooperative proton and electron fluxes. A model which combines the plasma membrane mechanism and the receptor-mediated endocytosis mechanism is presented.     

Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

Iron in evolution

Iron chemistry in the environment and in organisms is entwined. The iron surface minerals in solution for the first billion years of the planet were ferrous compounds. This ion became and has remained a major participant in organisms. The evolution of iron was due to its oxidation to insoluble ferric ions by oxygen released from organisms. The evolution of cellular iron chemistry then required uptake from this oxidised state. Use was expanded from the mainly electron transfer properties in the original reductive cell interior to employment in external oxidative chemistry. The environment/organisms evolution is that of one predictable chemical system.     

Redox properties of human transferrin bound to its receptor

Virtually all organisms require iron, and iron-dependent cells of vertebrates (and some more ancient species) depend on the Fe(3+)-binding protein of the circulation, transferrin, to meet their needs. In its iron-donating cycle, transferrin is first captured by the transferrin receptor on the cell membrane, and then internalized to a proton-pumping endosome where iron is released. Iron exits the endosome to enter the cytoplasm via the ferrous iron transporter DMT1, a molecule that accepts only Fe(2+), but the reduction potential of ferric iron in free transferrin at endosomal pH (approximately 5.6) is below -500 mV, too low for reduction by physiological agents such as the reduced pyridine nucleotides with reduction potentials of -284 mV. We now show that in its complex with the transferrin receptor, which persists throughout the transferrin-to-cell cycle of iron uptake, the potential is raised by more than 200 mV. Reductive release of iron from transferrin, which binds Fe(2+) very weakly, is therefore physiologically feasible, a further indication that the transferrin receptor is more than a passive conveyor of transferrin and its iron.     

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.     

Transport of ferrous iron and lactate production inBifidobacterium bifidum var.pennsylvanicus

Initial rates of ferrous iron transport intoBifidobacterium bifidum var.pennsylvanicus were measured at low and high iron concentrations. The low affinity system (LAFIUS) had an apparent K_m of 167 μM, the high affinity system (HAFIUS) had a K_m of 50 μM. Iron removal from preloaded bifidobacteria revealed the existence of a labile and an inert iron pool in the bacterial cells. Iron uptake by the bifidobacteria was associated with lactate production, though lactate production could continue without iron uptake. Cessation of iron uptake and lactate production was not because of an exhaustion of any nutrient nor the accumulation of fermentation end products in the medium. It was apparently the result of an inactivation of the cellular enzyme machinery without replacing it through normal biosynthetic processes.     

Dengue haemorrhagic fever and dengue shock syndrome: are they tumour necrosis factor-mediated disorders?

Growth of Bacteroides fragilis under anaerobic conditions in the presence of either haemin or protoporphyrin IX was inhibited by the ferrous iron chelator bipyridyl. The ferric-iron chelator desferrioxamine inhibited growth in the presence of protoporphyrin but not haemin, suggesting that even under anaerobic conditions Fe3+ is involved in uptake of non-haem iron, which is required in the absence of haemin. However, the ferric iron chelators 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and pyridoxal isonicotinoyl hydrazone (PIH) were only weakly inhibitory. Apotransferrin, which also binds Fe3+, inhibited growth, but this was not simply due to binding of iron in the medium, as under the reducing conditions present, transferrin was unable to bind iron. This study suggests that even under anaerobic conditions, uptake of non-haem iron by B. fragilis may involve conversion of Fe2+ to Fe3+.     

Separate pathways for cellular uptake of ferric and ferrous iron

Separate pathways for transport of nontransferrin ferric and ferrous iron into tissue cultured cells were demonstrated. Neither the ferric nor ferrous pathway was shared with either zinc or copper. Manganese shared the ferrous pathway but had no effect on cellular uptake of ferric iron. We postulate that ferric iron was transported into cells via beta(3)-integrin and mobilferrin (IMP), whereas ferrous iron uptake was facilitated by divalent metal transporter-1 (DMT-1; Nramp-2). These conclusions were documented by competitive inhibition studies, utilization of a beta(3)-integrin antibody that blocked uptake of ferric but not ferrous iron, development of an anti-DMT-1 antibody that blocked ferrous iron and manganese uptake but not ferric iron, transfection of DMT-1 DNA into tissue culture cells that showed enhanced uptake of ferrous iron and manganese but neither ferric iron nor zinc, hepatic metal concentrations in mk mice showing decreased iron and manganese but not zinc or copper, and data showing that the addition of reducing agents to tissue culture media altered iron binding to proteins of the IMP and DMT-1 pathways. Although these experiments show ferric and ferrous iron can enter cells via different pathways, they do not indicate which pathway is dominant in humans.