Cytosolic Ferritin Research Articles

Uptake of iron from transferrin by isolated hepatocytes. The effect of cellular energy metabolism on the intracellular distribution of iron and transferrin

The subcellular distribution of iron and transferrin has been studied in isolated rat hepatocytes during uptake of transferrin iron. Iron and transferrin are both rapidly transferred from an extracellular to an intracellular compartment in a process which is slowed down when the cells are deprived of ATP and completely blocked when the cells are incubated at 4 degrees C. The transfer of iron occurs at a higher rate than transferrin. The major part of iron is rapidly incorporated into cytosolic ferritin, i.e. after a 15-min incubation at 37 degrees C 60-70% of cell-associated iron is found in the cytosol as ferritin. The rest of the iron is found in mitochondria (5-10%) and, together with transferrin, in light and heavy endosomes. Following incubation at 4 degrees C, both iron and transferrin are confined to the plasma membrane whereas in ATP-depleted cells the majority of iron and transferrin are recovered in heavy endosomes. The results are consistent with receptor-mediated endocytosis as one mechanism for hepatocyte uptake of iron from transferrin but also suggest an alternative route by which transferrin can donate its iron to the cells and rapidly be released to the extracellular environment without undergoing a complete transferrin cycle.

Lipid associated tissue ferritin

Delipidation of liver homogenates, using an organic solvent system which does not denature proteins, increases measurable ferritin by 25–33%, compared to ferritin concentrations by standard heat supernatant assays. When applied to polyacrylamide gradient gels, lipid-associated ferritin does not enter the gel but after delipidation, this ferritin co-migrates with cytosolic ferritin and with purified liver ferritin. The biological significance of the association of ferritin with lipid has yet to be examined.

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.

Iron mobilization from cultured hepatocytes: Effect of desferrioxamine B

When cultured rat hepatocytes prelabelled for different times at 37° with 59Fe are reincubated for 1 hr in a fresh medium, radiolabelled iron is released in the washout medium as a function of the prelabelling time, and behaves like low molecular weight material on isokinetic centrifugation in sucrose gradients. When apotransferrin or desferrioxamine B are present in the reincubation medium, the kinetics of iron release are similar but the absolute amounts of radiolabelled iron found in the culture medium are much greater. In the presence of apotransferrin, most of the 59Fe released from the cells distributes as transferrin whereas with desferrioxamine B, almost all the 59Fe is extracted by benzyl alcohol indicating its chelation by the drug. Cell fractionation data indicate that iron accumulated by hepatocytes is rapidly incorporated into cytosol ferritin, and this seems to be a preferred source of iron for the chelator.

Iron utilization in rabbit reticulocytes: A study using succinylacetone as an inhibitor of heme synthesis

We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2- 14C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 μM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not δ-aminolevulinic acid, partly overcame the inhibition of 59Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits δ-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 μM, 0.1 and 1 mM, inhibited 59Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte 59Fe uptake by about 25–30%. In succinylacetone-treated cells 59Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and 59Fe-transferrin-pretreated reticulocytes results in the transfer of 59Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.

Cytosol intermediates in the transport of iron

Three 59Fe-labeled nonheme components of the cytosol were identified when rabbit reticuloyctes were incubated with 59Fe-labeled plasma under conditions in which the iron supply was not limiting. Two of these components were identified as ferritin and transferrin. The latter was characterized by gel filtration as having apparent molecular weight higher than transferrin, indicating that the transferrin may be complexed to another moiety. The third component, referred to as iron- binding protein-I (IBP-I), is as yet uncharacterized. When the reticulocytes were incubated with unlabeled plasma after pulse-labeling with 59Fe-labeled plasma, 59Fe radioactivity in these cytosol components decreased; after 15 min of chase, the 59Fe in ferritin, transferrin, and IBP-I fell to 64.6%, 26.5%, and 65.8% of the initial values, respectively. A good correlation existed between the decrease of 59Fe in these three nonheme compartments and the associated increase in 59Fe-heme. The data presented suggest that cytosol ferritin, transferrin, and IBP-I are intermediates in the transport of 59Fe from the plasma membrane to the mitochondria.

Cytosol Intermediates in the Transport of Iron

Three 59Fe-labeled nonheme components of the cytosol were identified when rabbit reticulocytes were incubated with 59Fe-labeled plasma under conditions in which the iron supply was not limiting. Two of these components were identified as ferritin and transferrin. The latter was characterized by gel filtration as having apparent molecular weight higher than transferrin, indicating that the transferrin may be complexed to another moiety. The third component, referred to as iron-binding protein-l (IBP-I), is as yet uncharacterized. When the reticulocytes were incubated with unlabeled plasma after pulse-labeling with 59Fe-labeled plasma, 59Fθ radioactivity in these cytosol components decreased; after 15 min of chase, the 59Fe in ferritin, transferrin, and IBP-I fell to 64.6%, 26.5%, and 65.8% of the initial values, respectively. A good correlation existed between the decrease of 59Fe in these three nonheme compartments and the associated increase in 59Fe-heme. The data presented suggest that cytosol ferritin, transferrin, and IBP-I are intermediates in the transport of 59Fe from the plasma membrane to the mitochondria.

Ferritin as a cytosol iron transport intermediate in human reticulocytes.

The major iron-bearing cytosol components of human reticulocytes identified after incubation with 59 Fe-125I-transferrin have been studied further. Component C previously found to behave consistently as an intermediate in the iron transport pathway to haem is shown to consist entirely of ferritin. After a short pulse of labelled transferrin incubation, chase experiments showed a fall of ferritin label with time and a corresponding increase in haemoglobin-iron incorporation. There was no loss of ferritin to the culture medium. Restriction of iron uptake by reticulocytes using both p-hydroxymercuribenzoate inhibition of uptake and incubation with progressively lower saturations of iron-transferrin gave linearly related incorporation of 59Fe into ferritin and haemoglobin at all levels of iron uptake, thus negating the concept of ferritin as an 'overspill' form of reticulocyte iron. The results suggest that cytosol ferritin is an obligatory intermediate in reticulocyte iron transport.

Iron uptake by Chang cells from transferrin, nitriloacetate and citrate complexes: The effects of iron-loading and chelation with desferrioxamine

Iron uptake by Chang liver cells in culture is about thirty times as great when ferric nitriloacetate is used as a donor as when iron-transferrin is used. Iron uptake from ferric citrate is no greater than from iron-transferrin. Most of the intracellular iron derived from transferrin is found in the supernatant after 20 000 × g centrifugation of the cell homogenate for 40 min: about half of this is in the form of ferritin. Iron derived from ferric nitriloacetate is found largley in the membranous pellet after centrifugation and very little of this is in the form of ferritin. Iron incorporated in cytosol ferritin is easily available for chelation by desferrioxamine and this process is facilitated by ascorbic acid. Membrane-bound iron is less available for chelation. This tissue culture model forms a convenient basis for the study of iron overlead and iron chelation.