Characterization of the binding of ferritin to the rat liver ferritin receptor

The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins.     

Studies on the mobilization of iron from ferritin by isolated rat liver mitochondria

Rat liver mitochondria and rat liver mitoplasts mobilize iron from ferritin by a mechanism which depends on a respiratory substrate (preferentially succinate), a small molecular weight electron mediator (FMN, phenazine methosulphate or methylene blue) and (near) anaerobic conditions.The release process under optimized conditions (approx. 50 μmol/l FMN, 1 mmol/l succinate, 0.35 mmol/l Fe(III) (as ferritin iron), 37°C and pH 7.40) amounts to 0.9–1.2 nmol iron/mg protein per min.The results suggest that ferritin might function as an intermediate in the cytosolic transport of iron to the mitochondria.     

The effect of iron on ferritin turnover in rat liver

125I-labelled angiotensin II (A II) specifically binds to solubilized receptors extracted from rat isolated glomeruli using CHAPS (3-[3-( cholamidopropyl ) dimethylammonio ]-1-propanesulfonate). The yield of solubilization of the binding sites was 3.3%. Equilibrium was reached after 15-20 min and specific binding represented 75% of total binding. Dissociation of the hormone-receptor complex after addition of an excess of A II was very slow in the presence of Ca2+ and Mg2+. [Sar1 Ala8] A II and [Sar1 Ile8] A II were more potent as competitive inhibitors of 125I-labelled A II than A II itself and its heptapeptide. These basic features of 125I-labelled A II binding to the extracted material were similar to those observed previously with untreated glomeruli.     

Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats

1.The content of non-heme iron and the degree of lipid peroxidation were measured in liver mitochondria isolated from rats injected with either Jectofer (an iron-sorbitol-citric acid complex) or iron-nitrilotriacetate. 2. The sedimentation profiles of the mitochondria from controls and iron-treated rats as revealed by analytical differential centrifugation, indicated single population of mitochondria with $\text{s}{}_{\mathrm{4,B}}$ values of 13200± 560 S and 14200±590 S for controls and iron-loaded animals, respectively. In contrast, the sedimentation profiles of the acid phosphatase activity and the non-heme iron revealed marked polydispersities with at least three populations of particles for both controls and iron-loaded animals. 3. The mitochondria and iron-rich lysosomes were separated by density-gradient centrifugation in an isotonic medium of Percoll and sucrose. With this technique, the amount of non-heme iron in a mitochondrial fraction by differential centrifugation decreased from 69±28 nmol/mg protein to 5.6±1.1 nmol/mg protein and from 19.3±5.6 nmol/mg protein to 3.3±0.6 nmol/mg protein for Jectofer and iron-nitrilotriacetate injected rats, respectively. For control rats the amount of mitochondrial non-heme iron was about 2.7 nmol/mg protein both before and following density gradient centrifugation. The extra amount of non-heme iron still present in the purified mitochondrial fraction from iron-loaded rats, as compared to controls, was further characterized by the reactivity towards bathophenanthroline sulfonate. The results suggest that the extra iron was due to a small amount of either ferritin or hemosiderin still contaminaning the mitochondrial fraction. The amount of mitochondrial heme iron was the same in iron-loaded rats and controls. 4. The degree of lipid peroxidation in the mitochondria was estimated from the amount of malondialdehyde. The thiobarbituric acid method used for the quantitation of malondialdehyde was modified so that it was insensitive to variable amounts of iron present in the samples. No difference in the degree of lipid peroxidation was observed between the mitochondria from iron-loaded rats and controls. 5. In contrast to recent proposals (Hanstein, E.G. et al. (1981) Biochim. Biophys. Acta 678, 293–299), the present study showed that the amounts of non-heme iron and the degrees of lipid peroxidation are the same in mitochondria isolated from iron-loaded and control animals.     

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.     

Serum ferritin: Past,present and future

Background

Serum ferritin was discovered in the 1930s, and was developed as a clinical test in the 1970s. Many diseases are associated with iron overload or iron deficiency. Serum ferritin is widely used in diagnosing and monitoring these diseases.

Scope of review

In this chapter, we discuss the role of serum ferritin in physiological and pathological processes and its use as a clinical tool.

Major conclusions

Although many aspects of the fundamental biology of serum ferritin remain surprisingly unclear, a growing number of roles have been attributed to extracellular ferritin, including newly described roles in iron delivery, angiogenesis, inflammation, immunity, signaling and cancer.

General significance

Serum ferritin remains a clinically useful tool. Further studies on the biology of this protein may provide new biological insights.     

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.     

Effect of phlebotomy on the ferritin iron content in the rat liver as determined morphometrically with the use of electron energy loss spectroscopy

Summary Phlebotomy of untreated and iron-loaded rats results in a significant decrease in total liver iron. In ironloaded rats a marked decrease in iron-containing particles is observed ultrastructurally in lysosomes and cytoplasm of hepatic sinusoidal cells but not in parenchymal cells. This remarkable phenomenon was further investigated in a morphometric study, based on element-specific (iron) distribution images made in situ in the parenchymal cell by means of electron energy loss spectroscopy. With the use of this technique it could be shown that in spite of phlebotomy the ferritin iron content of the iron-loaded liver parenchymal cell is not decreased.     

Effects of hexachlorobenzene and iron loading on rat liver mitochondria

The effects of hexachlorobenzene treatment and simultaneous iron-overload on the iron and porphyrin content of rat liver and rat liver mitochondria have been examined. In order to assess damages to the mitochondrial membrane occuring with these treatments, the content of malondialdehyde and selected functional properties of mitochondria were compared with those from control animals. Prolonged intake of hexachlorobenzene (8 weeks) resulted in a striking increased level of porphyrins together with a moderate increase in iron concentration. Simultaneous administration of hexachlorobenzene and iron-dextran caused the porphyrin level to reach 25% of the amount induced by hexachlorobenzene alone. The iron concentrations in liver as well as in liver mitochondria are also decreased under these conditions, as compared to the effect of iron-dextran. In contrast, the effects of hexachlorobenzene combined with iron-dextran on mitochondrial oxidative phosphorylation and malondialdehyde content are greater than those of either hexachlorobenzene or iron-dextran. These data suggest that porphyrin accumulation per se causes little deleterious effect and that both agents administered together act synergistically in causing damage to the mitochondrial membrane.     

Isolation of a ferritin from Bacteroides fragilis

A ferritin was isolated from the obligate anaerobe Bacteroides fragilis. Estimated molecular masses were 400 kDa for the holomer and 16.7 kDa for the subunits. A 30-residue N-terminal amino acid sequence was determined and found to resemble the sequences of other ferritins (human H-chain ferritin, 43% identity; Escherichia coli gen-165 product, 37% identity) and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 20% identity). The protein stained positively for iron, and incorporated 59Fe when B. fragilis was grown in the presence of [59Fe]citrate. However, the isolated protein contained only about three iron atoms per molecule, and contained no detectable haem. This represents the first isolation of a ferritin protein from bacteria. It may alleviate iron toxicity in the presence of oxygen.     

Magnetic properties of human liver and brain ferritin

Human brain (globus pallidus) and liver tissues were investigated by means of electron microscopy (EM), Mössbauer spectroscopy (MS) and SQUID magnetometry techniques. Based on MS measurements, the iron present was identified to be in the ferritin-like form (61–88%) and in the form of a low-spin iron species (the balance). Its overall concentration was estimated as 1.5(3) mg in the brain and 2.4(5) mg in the liver, per gram of lyophilized tissue. The average core diameter was determined by EM measurements to be equal to 7.5(1.3) nm for the liver and 3.3(5) nm for the brain. Magnetization measurements carried out between 5 and 300 K yielded an estimation of an average blocking temperature, KT _BL, as equal to 6.7 K and 8.5 K for the liver and the brain, respectively. From the dependence of KT _BL on the external magnetic field it was concluded that the ferritin-like cores in the studied samples can be regarded as non-interacting particles. Finally, the uniaxial magnetic anisotropy constant was determined to be 6×10³ J/m³ for the liver and 4×10⁴ J/m³ for the brain.     

Studies of ethylene-forming system in rat liver extract

Evidence of enzymatic formation of ethylene from methionine by rat liver extracts is presented. The ethylene production is closely associated with growth of the animal. The conversion of l-methionine to ethylene is oxygen dependent. Substrate analogue studies show that the ethylene-forming system is structurally specific and requires in the center of the molecule α-CH₂-CH₂- with one end attached to an unencumbered sulfur atom from a thioether moiety and the other end attached to a carboxyl group. Sylfhydryl agents are found to be very effective inhibitors of the ethylene-forming system. The finding of α-keto-4-methylthiobutyric acid to be a more efficient precursor of ethylene production suggests the possibility that α-keto-4-methylthiobutyric acid may be an intermediate in the biosynthesis of ethylene from methionine in mammalian tissues.     

Endocytosis of native and cationized ferritin by intralobular duct cells of the rat parotid gland

Summary The ability of the intralobular ducts of the rat parotid gland to take up protein from the lumen was examined after retrograde infusion of native and cationized ferritin. At high concentrations (3–10 mg/ml), cells of both intercalated- and striated ducts avidly internalized the tracers. No differences were noted in the mode of uptake or fate of native or cationized ferritin. Large, apical ferritin-containing vacuoles up to 5 m in size were present in cells of the intercalated ducts after infusion for 15 min. Small, smooth-surfaced spherical or flattened vesicles and tubules containing ferritin were also observed, often in association with the large vacuoles. Ferritin uptake increased with increasing infusion time, up to 1 h. Uptake by the striated ducts was less consistent than by the intercalated ducts, and occurred mainly in small vesicles and tubules. Secondary lysosomes became labeled with ferritin in both cell types. Ferritin was not observed in the Golgi saccules, nor was it discharged from the cells at the basolateral surfaces. At low concentrations (0.3–1 mg/ml), uptake was reduced, especially by cells of intercalated ducts, and differences were noted in the behavior of the two tracers. Cationized ferritin was internalized mainly into vesicles and tubules of cells of striated ducts; little uptake of native ferritin occurred at low concentrations. These results demonstrate that the ductal cells of the salivary glands are capable of luminal endocytosis of foreign proteins. They also suggest that in addition to modifying the primary saliva by electrolyte reabsorption and secretion, and secretion of various glycoproteins, the ductal cells are able to reabsorb proteins secreted by the acinar cells.     

Iron release from ferritin by flavin nucleotides

Background

Extensive in-vitro studies have focused on elucidating the mechanism of iron uptake and mineral core formation in ferritin. However, despite a plethora of studies attempting to characterize iron release under different experimental conditions, the in-vivo mobilization of iron from ferritin remains poorly understood.Several iron-reductive mobilization pathways have been proposed including, among others, flavin mononucleotides, ascorbate, glutathione, dithionite, and polyphenols. Here, we investigate the kinetics of iron release from ferritin by reduced flavin nucleotide, FMNH2, and discuss the physiological significance of this process in-vivo.

Methods

Iron release from horse spleen ferritin and recombinant human heteropolymer ferritin was followed by the change in optical density of the Fe(II)–bipyridine complex using a Cary 50 Bio UV–Vis spectrophotometer. Oxygen consumption curves were followed on a MI 730 Clark oxygen microelectrode.

Results

The reductive mobilization of iron from ferritin by the nonenzymatic FMN/NAD(P)H system is extremely slow in the presence of oxygen and might involve superoxide radicals, but not FMNH2. Under anaerobic conditions, a very rapid phase of iron mobilization by FMNH2 was observed.

Conclusions

Under normoxic conditions, FMNH2 alone might not be a physiologically significant contributor to iron release from ferritin.

General significance

There is no consensus on which iron release pathway is predominantly responsible for iron mobilization from ferritin under cellular conditions. While reduced flavin mononucleotide (FMNH2) is one likely candidate for in-vivo ferritin iron removal, its significance is confounded by the rapid oxidation of the latter by molecular oxygen.     

Cloning and characterization of Chlorobium tepidum ferritin

The Chlorobium tepidum ferritin (CtFtn) gene was synthesized and cloned into a pET3a expression vector (Novagen). CtFtn was expressed in Escherichia coli and purified to electrophoretic homogeneity. Sequence analysis indicates that all the conserved amino acids required to form the Fe²⁺ oxidizing ferroxidase center are present. Ftn is highly conserved from bacteria to humans, each subunit folds into a 4-helical bundle (helices A-D), with a long loop connecting helices B and C, plus a fifth short E-helix at the C-terminus. Calculations based on the secondary structure of CtFtn predict that each of these helices forms. However, the sequence of CtFtn shows a much longer C-terminus with a significant number of polar amino acids. Size-exclusion chromatography shows that CtFtn elutes at a size consistent with a 24-subunit protein cage. Incubation of CtFtn with Fe²⁺ produced an increase in the absorbance at 310 nm consistent with the incorporation of iron inside CtFtn. Assays monitoring ferroxidase activity showed that CtFtn possesses ferroxidase activity but it is less active than human H-chain ferritin. Additionally, the iron loading capacity of CtFtn is significantly reduced compared to proteins from other organisms. We propose that the unique extended C-terminus in CtFtn causes the decreased iron loading in CtFtn and possibly influences the slower rate of iron oxidation at the ferroxidase center.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Fungal ferritins: The ferritin from mycelia of Absidia spinosa is a bacterioferritin

Two distinct ferritin like iron containing proteins have been identified and isolated from the fungus Absidia spinosa; one from the spores and another from the mycelia. The mycelial protein has been purified and consists of two subunits of approx. 20 kDa. The N-terminal sequences of both subunits have been determined. The holoprotein as isolated contains approx. 750 iron atoms/molecule and exhibits a heme-like UV-Vis spectrum. Based on the heme spectrum and the high degree of sequence homology found, it has been established that the mycelial protein is a bacterioferritin. This is the first example demonstrating the presence of a bacterioferritin in a eukaryotic organism.     

Differential regulation of the two rice ferritin genes (OsFER1 and OsFER2)

Iron is essential to plants. However, when free and in excess, iron can catalyze the formation of oxygen free radicals. Ferritin, a protein capable of storing up to 4500 atoms of iron, can act as an iron buffer inside plant cells. Using a strategy based in amplicon size difference, we were able to analyze the expression profile of the two rice ferritin genes (OsFER1 and OsFER2). Both genes are expressed, although with different regulation and organ distribution. Exposure to copper, Paraquat, SNP and excess iron led to accumulation of ferritin mRNA, remarkably of OsFER2. The iron-induced expression was abolished by treatment with GSH, indicating that the induction observed is dependent of an oxidative step. OsFER2 mRNA levels in rice flag leaves and panicles at different reproductive stages were higher than OsFER1 mRNA levels. No ferritin mRNA was detected in rice seeds. However, imbibition under light led to ferritin expression, which was abolished when seeds were kept in the dark, suggesting a light-regulated induction. Ferritin mRNA accumulation was seen in the dark only when seeds were germinated in the presence of externally supplied iron. We suggest that the primary role of rice ferritins is related to defense against iron-mediated oxidative stress.     

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.