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.     

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.     

Structural differences in ferritins from normal and malignant rat tissues.

Ferritins purified from several normal and malignant rat tissues were examined for amino acid composition, content of tryptic peptides, available sulfhydryl groups and subunit sizes and proportion. Ferritin extracted from adult kidney, neonatal liver and hepatic and renal tumors differed from the ferritin of adult rat liver in migration on electrophoretic gels and in antibody affinity, but did not differ among themselves. Nevertheless, they showed distinctive differences in amino acid composition and tryptic peptide content. All of them and also adult liver ferritin contained two major species of subunits differing in molecular weight. The proportions of subunits, and the available sulfhydryl groups of the intact ferritin molecules, differed among these tissue ferritins. On the basis of amino acid and peptide content, the ferritins of hepatomas and the renal tumor analyzed showec the greatest similarity but not identity. The ferritin of neonatal liver was next most similar. Kidney ferritin differed considerably in composition from tumor and neonatal ferritins, while adult liver ferritin was the most extremely divergent of the series examined. A similar progressive difference was found on examining the proportions of subunits and sulfhydryl groups in these ferritins. However, changes in subunit proportion cannot explain the amino acid and peptide compositional changes.     

Translational regulation of ferritin synthesis in rat spleen: effects of iron and inflammation

Translational control of ferritin synthesis was studied in rat spleen, and compared with that for liver, heart and brain, in response to iron and inflammation. Spleen concentrations of total RNA in the ribonucleoprotein (mRNP) fraction was comparable to that for liver, while polyribosomal RNA was less. Both fractions were ten-fold lower in heart and brain. In untreated animals, the mRNP fraction of all tissues had the largest portion of the ferritin mRNA, as determined by slot blot hybridization with 32P-labeled cDNA for the L subunit. Acute treatment with ferric ammonium citrate shifted the spleen ferritin mRNA to the polyribosome fraction. This was also so in liver but not in the heart and brain which took up much less iron. The findings were confirmed by hybridization studies of mRNPs and polyribosomes separated in sucrose gradients. Turpentine-induced inflammation also caused a shift in ferritin mRNA from the mRNP to the polyribosome fraction of spleen and liver, over 12 h. We conclude that as in liver, spleen ferritin synthesis is under translational control by iron, and that both tissues also respond to inflammation by shifting of ferritin mRNA to the polyribosomes.     

Association of ferritin with liver cell membrane fractions.

Following intraperitoneal injection of rats with a large dose of ferric ammonium citrate containing ⁵⁹Fe, some 35 to 50% of the dose was deposited in the liver within the first 1 to 4 h. Almost all of the deposited iron could be precipitated with a ferritin antiserum from a homogenate of liver heated to 75 °C, but only half of this was precipitable when the unheated homogenate was treated with antiserum. The remainder of the ferritin iron was made available to the antiserum by treatment with deoxycholate, and was therefore presumed to be associated with membranous components of the cell.Subcellular fractionation of the liver following administration of ⁵⁹Fe-labeled ferric ammonium citrate showed that most of the radioactivity deposited within the first 4 h was equally divided between the cell sap and a light microsome (membrane-rich) fraction. Ferritin in this latter fraction was made available to antibody following deoxycholate treatment. The liver microsome fraction of the young rat contains little unavailable ferritin, but with aging there is an accumulation of ferritin in the microsomal fraction which is unavailable to antibody until the membrane is removed.It is suggested that at least part of the injected iron salt is taken up by pinocytotic vesicles and transferred to ferritin within this fraction, possibly followed by release of some of this ferritin into the cell sap.     

Effects of acute iron overload on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Distribution of radioiron to various tissues after intraperitoneal injections was examined in Atlantic salmon and rainbow trout. Liver and spleen were found to be the major iron storage tissues. Injections of 1 or 5 mg iron as ferric ammonium citrate led to a fall in hemoglobin levels in both species after 2 d. Hemoglobin levels returned to normal levels in rainbow trout after 8 d, but Atlantic salmon had not recovered, and Hb levels fell below 3 g/100 mL. In both species, the fall in Hb was associated with a raise in iron levels in spleen and liver, suggesting damage to erythrocytes. Atlantic salmon liver ferritin showed a two- to threefold increase, while rainbow trout showed a sixfold increase, and a more rapid response. The toxic effect of iron in fish appears to be different from the effect in other vertebrates.     

Relative distribution of arylsulphatases A and B in rat liver parenchymal and other cells

The relative activities of arylsulphatases A and B were measured in rat liver parenchymal and non-parenchymal cells, in peritoneal macrophages and in a number of rat tissues. Although absolute values cannot be obtained, it was shown that the arylsulphatase B/arylsulphatase A activity ratio is much higher in non-parenchymal cells than in parenchymal cells. The ratios in adrenals, brain and testis are very similar to each other but differ from those found in spleen, kidney and liver. These ratio variations may be caused by alterations in the activity of the B enzyme rather than the A enzyme. The relatively high B enzyme/A enzyme ratios in all rat tissues explains why the method devised for the independent assay of human arylsulphatases A and B cannot be employed with rat tissues.     

Size and charge heterogeneity of rat tissue ferritins.

Ferritins purified from horse spleen and from rat liver, kidney, heart and hepatoma were analyzed by quantitative polyacrylamide gel electrophoresis. From the migration characteristics of these ferritins at several gel concentrations, Ferguson plots were constructed and the molecular sizes and charges (apparent valences) together with their statistical variability were obtained by applying Rodbard computer programs to the data. Finally, ellipses were drawn describing the 95% confidence limits of these data for size and charge and were used to identify those ferritins that differed in size and/or charge. By these criteria, many of the tissue ferritins were differentiated from one another in terms of their molecular size and/or charge. Among the various tissue ferritin monomers, the molecular sizes were essentially similar (420 000-490 000) except for the two heart ferritins which were larger (530 000 and 626 000, respectively). However, the estimated charges on rat liver, kidney and hepatoma monomers (30-38 net protons per molecule) differed from that of spleen monomer (51 net protons per molecule) while the larger rat heart ferritin also had a greater charge (83 net protons) than the smaller (40 net protons). Apoferritins prepared chemically by removal of iron from the holoferritins had migration properties indistinguishable from the parent holoferritins. The migration properties of minor (dimeric) ferritin bands on the gels were compared with those of the monomer bands. The molecular sizes of the minor bands were larger than those of the major bands, and were not inconsistent with a doubling in size. However, charge differences varied, being either similar for major and minor forms (spleen ferritin), approximately twice for the minor form (rat hepatoma ferritin) or five times greater for the minor form (rat liver ferritin). These differences in behavior were confirmed by using minimally sieving gels, on which the major bands of horse spleen ferritin failed to separate whereas those of rat liver ferritin were readily separable. It is concluded that dimers of ferritins from different tissues may associate in different ways.     

Properties of human tissue isoferritins.

1. Human liver ferritin was separated by preparative isoelectric focusing into six fractions. 2. Except for the least acidic fraction the reactivity with antibody against spleen ferritin increased with rising pI, but with antibody against heart ferritin the reactivity decreased. 3. The highest iron content was found in the most acidic isoferritins and progressively decreased with rising pI. 4. Iron uptake was studied in apoferritin prepared from heart and liver ferritin fractions separated by ion-exchange chromatography. There was good correlation between the rate of iron uptake and pI. The most acidic fractions took up iron more rapidly than did the more basic ones. 5. Ferritin was prepared from heart, liver, spleen and kidney. There was little difference on isoelectric focusing between ferritin obtained from normal tissues and the corresponding iron-loaded tissues from patients who had received multiple blood transfusions. The iron-loaked heart ferritin invariably contained relatively more of the basic isoferritins. Normal and iron-overloaded heart ferritins were separated into isoferritin fractions by ion-exchange chromatography, and in each case there was a fall in iron content as the pI increased. The iron content of ferritin from the iron-overloaded heart was higher throughout than that from normal heart. 6. There is a relationship between the rate of iron uptake by apoferritin and pI, and this probably accounts for the variation in iron content of the isoferritins found in human liver and heart.     

Immunochemical characterization of human liver and heart ferritins with monoclonal antibodies

A library of 27 murine monoclonal antibodies was obtained by using human liver and heart ferritins as immunogens. The specificity of the antibodies for the two ferritins and their subunits was studied with five different methods. The antibodies elicited by the liver ferritin bound preferentially the immunogen and were specific for the L subunit. Some antibodies elicited by the heart ferritin had characteristics similar to the anti-liver antibodies, other ones bound preferentially the heart over the liver ferritin and were specific for the H subunit. Only two antibodies were able to bind both ferritins and subunits. Some anti-H and anti-L chain antibodies were used to develop and compare four types of immunoassay to quantitate isoferritins. The results indicate that heart ferritin is immunologically more heterogeneous than liver, the H and L subunits having large immunological differences with few, if any, identical epitopes; and that that the architecture of the immunoassays have a strong influence on the crossreactivity of the antibodies with the two isoferritins, probably because H and L chains are not arranged randomly in the assembled protein.     

Ferritin forms dynamic oligomers to associate with microtubules in vivo: implication for the role of microtubules in iron metabolism

Ferritin, a ubiquitously distributed iron storage protein, has been reported to interact with microtubules in vitro (Hasan et al., 2005, FEBS journal 272:822-831). Here, we demonstrate that ferritin binds with the microtubules in an oligomeric form and that the microtubule-bound ferritin contains more than two-fold amount of iron compared to the unbound ferritin fraction in vitro. Indirect immunofluorescence microscopy showed that a significant fraction of the ferritin molecules colocalized with the microtubules as oligomers in a wide variety of cell lines. These findings are consistent with the immediate oligomerization of rhodamine-labeled ferritin, microinjected in living human hepatoma cells. Ferritin oligomers were dynamic in the cytoplasm, and an anti-microtubule drug significantly inhibited their intracellular movement. Treatment of cells with an iron donor, ferric ammonium citrate, remarkably increased the number of cells containing ferritin oligomers. On the other hand, when the cells, such as mouse neuroblastoma cells, were deprived of iron, ferritin oligomers were localized in the microtubule dense, neurite shafts, but were disappeared from the microtubule deficient neurite tips. These data indicate that the microtubules provide a scaffold for the cytoplasmic distribution and transport of the iron-rich ferritin and implicate the role of microtubules in iron metabolism.     

Apolipoprotein B binds ferritin by hemin-mediated binding: evidence of direct binding of apolipoprotein B and ferritin to hemin

Apolipoprotein B (apoB) is known to be a ferritin-binding protein. Here we show that apoB binds to ferritin through hemin-mediated binding. Human apoB bound to bovine spleen, horse spleen, and canine liver ferritins, but did not bind to bovine apoferritin, even after incorporation of iron into it. Incubation of apoferritin with hemin resulted in apoB binding with apoferritin at the same level as with holoferritin. In contrast, hemin inhibited binding of apoB to ferritin. Bovine spleen apoferritin bound biotinylated hemin, and hemin inhibited the binding between the apoferritin and biotinylated hemin, suggesting that ferritin binds hemin directly. ApoB and LDL containing apoB bound biotinylated hemin, and their bindings were also inhibited by hemin, but not protoporphyrin IX. These data demonstrate that binding of apoB to ferritin is mediated through ferritin’s binding to hemin, and also that apoB binds hemin directly.     

An in vivo and in vitro comparison of the effects of amoxicillin,gentamicin, and cefazolin sodium antibiotics on the mouse hepatic and renal glutathione reductase enzyme

Despite the fact that the use of antibiotics is increasing worldwide, it is clear that antibiotics can lead to oxidative stress. This is the first study to make a comparison of the impact of frequently prescribed antibiotics, including amoxicillin, gentamicin, and cefazolin sodium, on the gene, protein, and activity of glutathione reductase (GR), which is one of the primary antioxidant enzymes, in mouse liver and kidney tissues. First, the GR enzyme was purified by the 2′,5′‐ADP Sepharose 4B affinity chromatography with a specific activity of 84.615 EU/mg protein and 9.63 EU/mg protein from the mouse liver and kidney, respectively. The in vitro inhibitory effects of the antibiotics in question was determined. While cefazolin sodium did not exhibit any inhibitory effect, gentamicin and amoxicillin inhibited GR activity in both tissues. Furthermore, the in vivo effects of these drugs were investigated, and amoxicillin and cefazolin sodium‐inhibited GR activity in both liver and kidney tissues, while gentamicin did not have any effect on the kidney. Besides, while gentamicin downregulated and cefazolin sodium upregulated Gr gene expression, amoxicillin did not alter it. Protein expression was only affected by the administration of cefazolin sodium in the kidney. This study is important as it demonstrates that while amoxicillin and gentamicin showed parallel effects on the GR activity in liver and kidney tissues both in vitro and in vivo, cefazolin sodium had a very strong effect on hepatic and renal GR in vivo. Furthermore, the antibiotics used in this study induced oxidative stress in both tissues.     

Characterization of ferritin from human placenta. Implications for analysis of tissue specificity and microheterogeneity of ferritins

Mammalian ferritins can be resolved into multiple components by isoelectric focusing, and each tissue contains a characteristic subset of isoferritins. Ferritin isolated from human liver was compared to acidic ferritin isolated from mid-gestational human placenta to define a structural basis for ferritin heterogeneity. Placenta ferritin contained several major bands with isoelectric points in the range of pI = 4.7-5.0 which were more acidic than the predominant isoferritins of human liver. Ferritin from each tissue was resistant to denaturation by 10 M urea and appeared to be identical by electron microscopy. Circular dichroism measurements revealed that placenta ferritin had substantially less ordered secondary structure than liver ferritin. Both types of ferritin contained only two subunits when analyzed by electrophoresis in sodium dodecyl sulfate gels, but isoelectric focusing of dissociated subunits in urea revealed 6-7 different components. In this system, placenta ferritin was enriched in the more acidic subunits and it completely lacked the most basic subunits noted in liver ferritin; placental ferritin had no unique components. Differences in isoelectric points among assembled ferritins from these two tissues appear to result from different proportions of these acidic and basic subunits.     

单抗在人铁蛋白免疫亲和纯化中的应用

抗人铁蛋白单抗６Ｄ６和Ａ－ｈＦ－Ｃ与肝型铁蛋白和心型铁蛋白的反应性有所不同。６Ｄ６对两种铁蛋白的反应性相似;而Ａ－ｈＦ－Ｃ单抗与肝型铁蛋白的反应性较强。我们将６Ｄ６和Ａ－ｈＦ－Ｃ分别制成了亲和凝胶,用来纯化人肝脏和心脏粗抽提物中的铁蛋白。此法具有操作简便,产率和产品纯度高等优点。     

Heterogeneity in tissue ferritins displayed by gel electrofocusing

1. Horse spleen ferritin and human liver ferritin were examined by gel electrofocusing under conditions that demonstrated equilibrium focusing. Both ferritins were resolved into multiple isoferritins. Both families of isoferritins were separable from one another. 2. Horse spleen ferritin was also resolved into five components by ion-exchange chromatography on DEAE-Sephadex A-50. Each of the major chromatographic fractions contained only a few of the isoferritins seen on gel electrofocusing. Each chromatographic fraction corresponded to different portions of the isoferritin profile. 3. These results indicate that the heterogeneity seen in many ferritins by gel electrofocusing represents structural heterogeneity in the ferritin population as isolated from the tissues.     

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.     

Control of ketogenesis from amino acids. IV. Tissue specificity in oxidation of leucine, tyrosine, and lysine.

In vitro and in vivo studies were made on the tissue specificity of oxidation of the ketogenic amino acids, leucine, tyrosine, and lysine. In in vitro studies the abilities of slices of various tissues of rats to form 14CO2 from 14C-amino acids were examined. With liver, but not kidney slices, addition of alpha-ketoglutarate was required for the maximum activities with these amino acids. Among the various tissues tested, kidney had the highest activity for lysine oxidation, followed by liver; other tissues showed very low activity. Kidney also had the highest activity for leucine oxidation, followed by diaphragm; liver and adipose tissue had lower activities. Liver had the highest activity for tyrosine oxidation, but kidney also showed considerable activity; other tissues had negligible activity. In in vivo studies the blood flow through the liver or kidney was stopped by ligation of the blood vessels. Then labeled amino acids were injected and recovery of radioactivity in respiratory 14CO2 was measured. In contrast to results with slices, no difference was found in the respiratory 14CO2 when the renal blood vessels were or were not ligated. On the contrary ligation of the hepatic vessels suppressed the oxidations of lysine and tyrosine completely and that of leucine partially. Thus in vivo, lysine and tyrosine seem to be metabolized mainly in the liver, whereas leucine is metabolized mostly in extrahepatic tissues and partly in liver. Use of tissue slices seems to be of only limited value in elucidating the metabolisms of these amino acids.     

Ferritins can regulate the secretion of apolipoprotein B

Apolipoprotein B is secreted with atherogenic lipids as lipoprotein particles from hepatocytes. Regulation of the secretion of apolipoprotein B is largely post-translational and reflects the balance between processes that leads to particle assembly or to intracellular degradation. Previously, we conducted a proteomic screen to find proteins that bind apolipoprotein B in rat liver microsomes. We identified ferritin heavy and light chains in this screen among other proteins and showed that the two ferritins bind apolipoprotein B directly in vitro. In hepatocytes and other cells, ferritin heavy and light chains form cytosolic cages that store iron. We now show that ferritin heavy or light chains post-translationally inhibit the secretion of apolipoprotein B without altering the export of other hepatic proteins including albumin, factor XIII, and apolipoprotein A-I. This inhibition of apolipoprotein B secretion is not due to diminished lipid synthesis and can be partially overcome by stimulating triglyceride synthesis. The block in apolipoprotein B secretion by ferritins leads to an increase in endoplasmic reticulum-associated degradation of the apolipoprotein. Thus, despite being cytosolic proteins without known chaperone activity, ferritins can specifically regulate the secretion of apolipoprotein B post-translationally. The metabolic pathways for iron storage and intercellular cholesterol and triglyceride transport could intersect.