Quantitative analysis of immunogold labelling for ferritin in liver from control and iron-overloaded rats

Summary The distribution of ferritin antigenicity in control and iron-loaded rat hepatocytes was investigated with an immunogold-ferritin antibody technique. Antibody to horse spleen ferritin showed immunoreactivity as determined by dot blotting with immunogold/silver staining with purified rat liver ferritin but not with rat haemosiderin. The initial site of ferritin degradation was studied by analysing the density of gold labelling in the cytosol and lysosomes in combination with pre-embedding acid phosphatase cytochemistry.Immunoreactive ferritin was present in the cytosol, cytosolic clusters and lysosomes of normal hepatocytes. After iron-loading, the labelling density increased over tenfold in parenchymal cell cytosol with a smaller increase in Kupffer cells. Ferritin clusters contained substantially more immunoreactive ferritin than equivalent areas of lysosomes or cytosol. Analysis of the labelling density in hepatocyte lysosomes showed that, despite a striking increase in iron content, one-quarter of the lysosomes showed less immunolabelled ferritin than the cytosol. The existence of a wide range of ferritin labelling densities in the lysosomes with a large proportion unlabelled suggests that the ferritin protein shell is not degraded at a significant rate either in the cytosol or in clusters but only after incorporation into lysosomes.     

Purification and characterization of a small dermatan sulphate proteoglycan implicated in the dilatation of the rat uterine cervix.

Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results were consistent with the difference in calculated isoelectric points of chicken and horse ferritin subunits. By two-dimensional gel electrophoresis, chicken ferritin 22 kDa subunits exhibited isoelectric points from 6.1 to 6.6 whereas horse spleen ferritin subunits exhibited isoelectric points of 5.8-6.3. The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6 whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9. Intact chicken liver ferritin particles were 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in diameter when viewed by electron microscopy. Horse spleen ferritin consisted of slightly smaller particles with an average diameter of 11.0 +/- 0.7 nm. However, ferritin from chicken liver and horse spleen co-migrated with an apparent molecular mass of 470 kDa when analysed by Sepharose 4B gel filtration chromatography. These results indicate that, consistent with results from other published purification methods, the chicken ferritin purified by the methods reported here exhibits both structural similarities to, and differences from, horse spleen ferritin.     

In vivo endocytosis by bristle coated pits of protein tracers and their intracellular transport in the endothelial cells lining the sinuses of the liver. II. The endosomal-lysosomal transformation

The events leading to lysosomal activity in the sinus endothelium of the rat liver have been studied by means of intravascularly injected ferritin at time intervals ranging from 0.5 min to 1 hr after administration. From 6 min on, the dense body-type lysosomes contain ferritin. There are direct luminal communications of transfer tubules with these lysosomes. In time, there is a marked progressive increase in the number of ferritin-containing dense body-type lysosomes. No formation of lysosomes de novo nor a direct fusion of endosomes with lysosomes has been observed. Endosomes, however, continue to be formed as endocytosis continues. These observations are interpreted as indicating a transport of hydrolytic enzymes by the transfer tubules to the newly formed ferritin containing endosomes, which in this way are transformed into ferritin containing lysosomes. The ferritin-containing lysosomes increase considerably in size by fusing with each other. Continued endocytosis of ferritin leads to an increase of ferritin density in the dense bodies. This increase in particle density cannot be explained solely on the basis of transport by luminal fusion of the endocytic organelles, but requires an active transport mechanism. Administration of low doses of ferritin shows that the bristle coated pits of the sinus endothelium have a high degree of in vivo affinity for protein and that this endothelium must be considered to be an avid catabolic endocytic system.     

Lysosomal and cytosolic ferritins A biochemical and electron-spectroscopic study

Summary Cytosolic and lysosomal ferritin and haemosiderin were isolated from rat livers which had been iron-loaded by four intraperitoneal injections of iron-dextran. The cytosolic and lysosomal ferritins, prepared in a phosphate-free medium, were subjected to gel-filtration chromatography on Sepharose 613, yielding four fractions: a cytosolic monomeric (CMF) and void-volume ferritin fraction (CVVF), and a lysosomal monomeric (LMF) and void-volume ferritin fraction (LVVF). Of each fraction the following aspects were examined: (a) immunoreactivity against specific antiserum; (b) the Fe/P mass ratio and the effect of dialysis on this ratio using electron probe micro-analysis (EPMA); (c) morphology and Fespecific imaging using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). For haemosiderin one aspect, the Fe/P ratio, was determined before and after extensive purification. The following results were obtained (a) All ferritin fractions reacted with anti- (rat liver ferritin). (b) The Fe/P ratios as determined in CMF in an haemosiderin were not affected by dialysis or extensive purification, respectively. The Fe/P ratio in CWF was affected by dialysis. In the lysosomal fractions, only a trace of phosphorus (LVVF) or no phosphorus (LMF) was detected. (c) Morphologically, CMF and CVVF were found to be rather homogeneous; the iron core diameters of both fractions were in the known size range. LMF and LVVF were of rather heterogeneous composition; the core diameters of these fractions were different. In conclusion: the phosphorus in ferritin and haemosiderin is firmly bound; Haemosiderin, when derived from ferritin, has to take up phosphorus in the lysosomes.     

The monomers and oligomers of ferritin and apoferritin: association and dissociation.

We have reinvestigated the association and dissociation of ferritin and apoferritin in phosphate buffer (pH 7.2, I = 0.05). When oligomer-enriched solutions of horse spleen ferritin were mixed with more concentrated, but unenriched solutions of horse spleen apoferritin, there was dissociation of the ferritin oligomers, as determined by polyacrylamide gel electrophoresis and from iron/protein ratios. Some evidence was also obtained for association of monomers in the mixture of ferritin and apoferritin after pelleting and redissolution of pellets in minimal volumes of the phosphate buffer. Monomer-enriched, biosynthetically labeled rat liver ferritin was pelleted, redissolved in minimal volumes of phosphate buffer, and separated by polyacrylamide gel electrophoresis; the fractions were isolated and counted. The results revealed that an association of monomers of the rat liver ferritin had taken place which doubled the concentration of dimers. However, our results also indicate that association by concentration was limited to a fraction of monomers.     

Iron (III) can be transferred between ferritin molecules.

The iron-storage molecule ferritin can sequester up to 4500 Fe atoms as the mineral ferrihydrite. The iron-core is gradually built up when FeII is added to apoferritin and allowed to oxidize. Here we present evidence, from M?ssbauer spectroscopic measurements, for the surprising result that iron atoms that are not incorporated into mature ferrihydrite particles, can be transferred between molecules. Experiments were done with both horse spleen ferritin and recombinant human ferritin. M?ssbauer spectroscopy responds only to 57Fe and not to 56Fe and can distinguish chemically different species of iron. In our experiments a small number of 57FeII atoms were added to two equivalent apoferritin solutions and allowed to oxidize (1-5 min or 6 h). Either ferritin containing a small iron-core composed of 56Fe, or an equal volume of NaCl solution, was added and the mixture frozen in liquid nitrogen to stop the reaction at a chosen time. Spectra of the ferritin solution to which only NaCl was added showed a mixture of species including 57FeIII in solitary and dinuclear sites. In the samples to which 150 56FeIII-ferritin had been added the spectra showed that all, or almost all, of the 57FeIII was in large clusters. In these solutions 57FeIII initially present as intermediate species must have migrated to molecules containing large clusters. Such migration must now be taken into account in any model of ferritin iron-core formation.     

Mobilization of iron from ferritin by isolated mitochondria effects of species compatibility between ferritin and mitochondria and iron content of ferritin

Mitochondria mobilize iron from ferritin by a mechanism that depends on external FMN. With rat liver mitochondria, the rate of mobilization of iron is higher from rat liver ferritin than from horse spleen ferritin. With horse liver mitochondria, the rate of iron mobilization is higher from horse spleen ferritin than from rat liver ferritin. The results are explained by a higher affinity between mitochondria and ferritins of the same species. The mobilization of iron increases with the iron content of the ferritin and then levels off. A maximum is reached with ferritins containing about 1 200 iron atoms per molecule. The results represent further evidence that ferritin may function as a direct iron donor to the mitochondria.     

Characterization of the H- and L-subunit ratios of ferritins by sodium dodecyl sulfate-capillary gel electrophoresis

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was used to characterize the H- and L-subunit ratios of several mammalian ferritins and one bacterioferritin. Traditionally, SDS-PAGE has been used to characterize the H- and L-subunit ratios in ferritin; however, this technique is relatively slow and requires staining, destaining, and scanning before the data can be processed. In addition, the H- and L-subunits of ferritin are fairly close in molecular weight (approximately 21,000 and approximately 20,000, respectively) and are often difficult to resolve in SDS-PAGE slab gels. In contrast, SDS-CGE requires no staining or destaining procedures and the peak quantitation is superior to SDS-PAGE. SDS-CGE is effective in quickly resolving the H- and L-subunits of ferritins from horse spleen, human liver, recombinant human H and L homopolymers, and mixtures of the two- and the single-subunit of a bacterioferritin from Escherichia coli. The technique has also proven useful in assaying the quality of the protein sample from both commercial and recombinant sources. Significant amounts of low-molecular-weight degradation products were detected in all commercial sources of horse spleen ferritin. Most commercial horse spleen ferritins lacked intact H-subunits under denaturing conditions.     

铁核结构对马脾铁蛋白释放铁动力学的影响

建立Ｈ^% 参与马脾铁蛋白释放铁的动力方程,Ｈ^ 以１／２级反应方式参与铁蛋白释放铁核表层的铁。在酸性介质（ＰＨ６．５）中,铁蛋白释放铁的总平均速率（３３２Ｆe^3 /HSF.min)比在碱性介质（Ｐ8Ｈ８．０）中放铁的总平均速率（７３Ｆe^3 /HSF.min)高４．６倍,铁蛋白的铁核结构和外加的磷酸盐均能影响该蛋白释放的速率,但并不改变其反应级数。     

Analysis of iron-containing compounds in different compartments of the rat liver after iron loading

Summary The livers of iron-loaded rats were fractionated and a cytosolic fraction, a lysosomal fraction, a siderosomal fraction and haemosiderin were obtained. All iron-containing compounds from these fractions were isolated and their morphology, Fe/P ratios, iron core diameter and peptide content were compared. The cytosolic fraction contained ferritin (CF) and a slower sedimenting, light ferritin (CLF). The lysosomal fraction also contained ferritin (LF) and a slower sedimenting light ferritin (LLF). The siderosomal fraction contained ferritin (SF), a faster sedimenting non-ferritin iron compound (SIC) and haemosiderin (HS). SIC and HS did not resemble ferritin as much as the other products did, but were found to be water-insoluble aggregates. The Fe/P ratios of CF and CLF were lower than the Fe/P ratios of LF and LLF and these in turn had lower Fe/P ratios than SF, SIC and HS. The iron core diameter of the cytosolic ferritin was increased after lysosomal uptake. The iron core diameters of the siderosomal products were smaller. CLF, CF, LF, LLF and SF contained one kind of subunit of approximately 20.5 kDa. SIC and HS contained other peptides in addition to the 20.5-kDa subunit. The results indicate that storage of ferritin molecules is not limited to the cytosolic compartment, but is also the case in the lysosomes. Extensive degradation of the ferritin molecule seems to be confined to the siderosomes.     

The ferroxidase center is essential for ferritin iron loading in the presence of phosphate and minimizes side reactions that form Fe(III)-phosphate colloids

Ferritin iron loading was studied in the presence of physiological serum phosphate concentrations (1 mM), elevated serum concentrations (2–5 mM), and intracellular phosphate concentrations (10 mM). Experiments compared iron loading into homopolymers of H and L ferritin with horse spleen ferritin. Prior to studying the reactions with ferritin, a series of control reactions were performed to study the solution chemistry of Fe²⁺ and phosphate. In the absence of ferritin, phosphate catalyzed Fe²⁺ oxidation and formed soluble polymeric Fe(III)-phosphate complexes. The Fe(III)-phosphate complexes were characterized by electron microscopy and atomic force microscopy, which revealed spherical nanoparticles with diameters of 10–20 nm. The soluble Fe(III)-phosphate complexes also formed as competing reactions during iron loading into ferritin. Elemental analysis on ferritin samples separated from the Fe(III)-phosphate complexes showed that as the phosphate concentration increased, the iron loading into horse ferritin decreased. The composition of the mineral that does form inside horse ferritin has a higher iron/phosphate ratio (~1:1) than ferritin purified from tissue (~10:1). Phosphate significantly inhibited iron loading into L ferritin, due to the lack of the ferroxidase center in this homopolymer. Spectrophotometric assays of iron loading into H ferritin showed identical iron loading curves in the presence of phosphate, indicating that the ferroxidase center of H ferritin efficiently competes with phosphate for the binding and oxidation of Fe²⁺. Additional studies demonstrated that H ferritin ferroxidase activity could be used to oxidize Fe²⁺ and facilitate the transfer of the Fe³⁺ into apo transferrin in the presence of phosphate.     

M?ssbauer spectroscopic investigation of structure-function relations in ferritins.

Ferritin plays an important role in iron metabolism and our aim is to understand the mechanisms by which iron is sequestered within its protein shell as the mineral ferrihydrite. We present M?ssbauer spectroscopic data on recombinant human and horse spleen ferritin from which we draw the following conclusions: (1) that apoferritin catalyses Fe(II) oxidation as a first step in ferrihydrite deposition, (2) that the catalysis of Fe(II) oxidation is associated with residues situated within H chains, at the postulated 'ferroxidase centre' and not in the 3-fold inter-subunit channels previously suggested as the initial Fe(II) binding and oxidation site; (3) that both isolated Fe(III) and Fe(III) mu-oxo-bridged dimers found previously by M?ssbauer spectroscopy to be intermediates in iron-core formation in horse spleen ferritin, are located on H chains; and (4) that these dimers form at ferroxidase centres. The importance of the ferroxidase centre is suggested by the conservation of its ligands in many ferritins from vertebrates, invertebrates and plants. Nevertheless iron-core formation does occur in those ferritins that lack ferroxidase centres even though the initial Fe(II) oxidation is relatively slow. We compare the early stages of core formation in such variants and in horse spleen ferritin in which only 10-15% of its chains are of the H type. We discuss our findings in relation to the physiological role of isoferritins in iron storage processes.     

Heterogeneity in horse ferritins. A comparative study of surface charge, iron content and kinetics of iron uptake.

Horse ferritins from different organs show heterogeneity on electrofocusing in Ampholine gradients. Both ferritin and apoferritin from liver and spleen could be fractionated with respect to surface charge by serial precipitation with (NH4)2SO4. In the ferritin fractions, increasing iron content parallels increasing isoelectric point. After removal of their iron, those fractions which originally contained most iron accumulated added iron at the fastest rates. When unfractionated ferritins from different organs were compared the average isoelectric point increased in order spleen less than liver less than kidney less than heart. The order of initial rates of iron uptake by the apoferritins was spleen greater than kidney greater than heart and initial average iron contents also followed this order. The relatively low rates of iron accumulation by iron-poor molecules may have been due to structural alteration, to degradation, to activation of the iron-rich molecules or to other factors.     

Heart tissue contains small and large aggregates of ferritin subunits

Ferritin purified from horse heart and applied to nondenaturing polyacrylamide gel electrophoresis migrated as a single band that stained for both iron and protein. This ferritin contained almost equal amounts of fast- and slow-sedimenting components of 58 S and 3-7 S, which could be separated on sucrose density gradients. Iron removal reduced the sedimentation coefficient of the fast-sedimenting ferritin to 18 S, and sedimentation equilibrium gave a molecular weight 650,000, with some preparations containing ferritin of 500,000 molecular weight as well. Sedimentation rates of the 3 S and 7 S ferritins were not affected by iron removal, and sedimentation equilibrium data were consistent with Mr's 40,000 and 180,000, respectively. Preparations of ferritin extracted from horse spleen contained only 67 S (holo) or 16 S (apo) ferritin and no slow-sedimenting species. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all of the ferritins contained the usual H and L subunits (23 and 20 kDa, respectively), but the slow-sedimenting (3 S and 7 S) heart apoferritins also contained appreciable quantities (ca 25%) of three larger subunits of 42, 55, and 65 kDa. All the subunits reacted positively in Western blots to polyclonal antibodies made against specially purified large heart or spleen ferritins containing only 20- and 23-kDa subunits. Similar results were obtained for ferritins from rat heart. The results indicate that mammalian heart tissue is peculiar not just in having an abnormally large iron-rich ferritin but also in having iron-poor ferritins of much lower molecular weight, partly composed of larger subunits.     

Purification and characterization of ferritin fromCampylobacter jejuni

We purified an iron-containing protein from Campylobacter jejuni using ultracentrifugation and ion-exchange chromatography. Electron microscopy of this protein revealed circular particles with a diameter of 11.5 nm and a central core with a diameter of 5.5 nm. The protein was composed of a single peptide of 21 kDa and did not serologically cross-react with horse spleen ferritin. The UV-visible spectrum of the protein showed no absorption peaks in the visible region, indicating that little or no heme is bound. The ratio of Fe:phosphate of C. jejuni ferritin was 1.5:1. From these morphological and chemical examinations, we concluded that the C. jejuni purified protein is a ferritin of the same class as that of Helicobacter pylori and Bacteroides fragilis and differs from the heme-containing bacterioferritin of Escherichia coli. The 30 N-terminal amino acids were sequenced and were found to resemble the sequences of other ferritins strongly (H. pylori ferritin, 73% identity; B. fragilis ferritin, 50% identity; E. coli gene-165 product, 50% identity), and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 26% identity; Azotobacter vinelandii, 26% identity; horse spleen ferritin 30% identity). Proteins that cross-reacted with antiserum against the ferritin of C. jejuni were found in other Campylobacter species and in H. pylori, but not in Vibrio, E. coli, or Pseudomonas aeruginosa. Received: 6 September 1994 / Accepted: 6 February 1995     

Isolation and characterization of Phycomyces blakesleeanus ferritin.

Ferritin was isolated from the fungus Phycomyces blakesleeanus and compared biochemically and immunologically with horse spleen ferritin. Phycomyces and horse spleen ferritins were shown to exhibit similar electrophoretic patterns on polyacrylamide gels. Both preparations yielded an identical single band on sodium dodecyl sulfate-containing polyacrylamide gels. Tryptic digests of Phycomyces ferritin yielded 17 ninhydrin-positive spots as compared to 26 for horse spleen ferritin tryptic digests. Phycomyces ferritin was immunologically unrelated to horse spleen ferritin.     

Characterization of a ferritin isolated from the midgut epithelial cells of a homopteran insect, Philaenus spumarius L

Crystalline accumulations of ferritin-like particles are present within the cytoplasma and the nucleus in midgut epithelial cells of the homopteran Philaenus spumarius. A structural study at the electron microscope level reveals that these particles have the morphological characteristics of the ferritin molecule: crystals have a face-centered cubic structure with a lattice parameter of 14 +/- 1 nm; negatively stained isolated particles have the appearance of ferritin; on rotary-shadowed particles 3 axes of symmetry are clearly seen; image processing performed on selected molecules demonstrates a 4-fold symmetry. A semiquantitative electron microprobe analysis effected on aggregates of microcrystals in thin sections reveals a high atomic ratio Fe/P. Analyzed by SDS-PAGE, the protein subunit has a molecular weight of 18,600. The amino acid composition of the protein bears the general characteristics of the ferritin molecule in terms of polar and nonpolar residues. But in terms of sequences, this protein displays a strong dissimilarity to rat liver ferritin as demonstrated with a common amino acid index test and with immunoelectrophoresis experiments.     

Studies on ferritin in rat liver and spleen during repeated phlebotomy

1. The ferritin content of liver and spleen in normal and iron-loaded rats decreased during repeated phlebotomy. 2. During increased iron demand, ferritin is degraded in toto. 3. With the ESI and EELS technique the iron distribution was followed in different cell types and cellular compartments. 4. We have demonstrated two methods of iron mobilisation: (a) catabolism of lysosomal ferritin in toto and (b) delivery of ferritin from parenchymal cell into the bile and degradation of ferritin in toto.     

Properties and role of ferritin in the hemolymph of the chiton Clavarizona hirtosa

The major iron-binding protein found in the hemolymph of the chiton Clavarizona hirtosa has been purified for the first time and identified as ferritin. This ferritin, which is present at a concentration of approx. 400 μg·ml⁻¹, has a M_r of 28 000 and 25 500, exhibits microheterogeneity with isoelectric values in the range 5.3–6.0, binds 1500–2500 Fe atoms·mol⁻¹ and is immunologically distinct from horse spleen ferritin. The initial rate of iron accumulation by ferritin molecules was determined to be markedly higher than that exhibited by horse spleen ferritin. Taken together, these data suggest that ferritin found in the hemolymph serves as a key component of the high-capacity transport system necessary to deliver iron to the rapidly mineralizing tissue of the radula in these molluscs.     

Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe2+ to O2 stoichiometry of iron deposition in horse spleen ferritin

Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition. Stoichiometric amounts of H2O2 have not been measured, and H2O2 is proposed to react with an unknown system component. Evidence is presented that identifies this component as amine buffers, including 3-N-morpholinopropanesulfonic acid (MOPS), which is widely used in ferritin studies. In the presence of non-amine buffers, the Fe2+ to O2 stoichiometry was approximately 4.0, but at high concentrations of amine buffers (0.10 M) the Fe2+ to O2 stoichiometry is approximately 2.5 for iron loadings of eight to 30 Fe2+ per HoSF. Decreasing the concentration of amine buffer to zero resulted in an Fe2+ to O2 stoichiometry of approximately 4. Direct evidence for amine buffer modification during Fe2+ deposition was obtained by comparing authentic and modified buffers using mass spectrometry, NMR, and thin layer chromatography. Tris(hydroxymethyl)aminomethane, MOPS, and N-methylmorpholine (a MOPS analog) were all rapidly chemically modified during Fe2+ deposition to form N-oxides. Under identical conditions no modification was detected when amine buffer, H2O2, and O2 were combined with Fe2+ or ferritin separately. Thus, a short-lived ferritin intermediate is required for buffer modification by H2O2. Variation of the Fe2+ to O2 stoichiometry versus the Fe2+ to HoSF ratio and the amine buffer concentration are consistent with buffer modification.