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1.
Murine erythroleukaemic cells were studied to determine whether different isoferritins have different functions. The cells were labelled with radioactive iron and the pattern of isoferritins was analysed by chromatofocussing. No change was found after iron-loading the cells but after inducing erythroid differentiation with dimethyl sulphoxide (DMSO), iron was incorporated into both more basic and more acidic isoferritins. This was compared to ferritin subunit synthesis; DMSO induced the synthesis of a third, minor subunit whereas iron-loading had no effect. The fate of murine erythroleukaemic cell ferritin iron was followed after incubations in iron-deficient medium containing DMSO; some, but not all, of the ferritin iron was mobilized and used for haem synthesis, and the remaining iron was found amongst the more basic isoferritins. Finally, sequential radioactive iron labels were used to demonstrate that the movement of iron from ferritin to haem was compatible with the 'last-in-first-out' principle, but this could not be related to different isoferritins. These results show firstly that DMSO changes the pattern of isoferritins and ferritin subunits in murine erythroleukaemic cells. Secondly, iron associated with more basic isoferritins seems to be less easily mobilized for haem synthesis. These results support the concept that different isoferritins have different functions.  相似文献   

2.
Murine erythroleukaemic cells were studied to determine whether different isoferritins have different functions. The cells were labelled with radioactive iron and the pattern of isoferritins was analysed by chromatofocussing. No changes was found after iron-loading the cells but after inducing erythroid differentiation with dimethyl sulphoxide (DMSO), iron was incorporated into both more basic and more acidic isoferritins. This was compared to ferritin subunit synthesis; DMSO induced the synthesis of a third, minor subunit whereas iron-loading had no effect. The fate of murine erythroleukaemic cell ferritin iron was followed after incubations in iron-deficient medium containing DMSO; some, but not all, of the ferritin iron was mobilized and used for haem synthesis, and the remaining iron was found amongst the more basic isoferritins. Finally, sequential radioactive iron labels were used to demonstrate that the movement of iron from ferritin to haem was compatible with the ‘last-in-first-out’ principle, but this could not be related to different isoferritins. These results show firstly that DMSO changes the pattern of isoferritins and ferritin subunits in murine erythroleukaemic cells. Secondly, iron associated with more basic isoferritins seems to be less easily mobilized for haem synthesis. These results support the concept that different isoferritins have different functions.  相似文献   

3.
Functional studies on rat-liver isoferritins   总被引:1,自引:0,他引:1  
Rat-liver and horse-spleen isoferritins were obtained by preparative isoelectric focussing and several of these were fractionated further by sucrose density gradient centrifugation. H- and L-subunit compositions were also measured. Isoferritins were found to have neither a fixed iron content nor a unique subunit composition. In both species within a single isoferritin a small increase in the percentage of H subunit paralleled increasing iron content. Although in horse-spleen ferritin a similar correlation was found over the isoferritin profile as a whole, this was not generally true of rat-liver isoferritins, since iron distributions varied with the iron status of the animals. Rates of iron incorporation into isoapoferritins were measured in vitro and the distribution of 59Fe among rat liver isoferritins was measured at various times after injection of 59Fe. The data do not support the proposal that, in rat liver, L-rich isoferritins are the preferred iron-storage form.  相似文献   

4.
Ferritin-binding proteins circulating in mammalian blood are thought to be involved in the clearance of ferritin. The present study characterizes canine serum autoantibodies (IgM and IgA) that react with ferritin. Canine IgM and IgA bound to bovine spleen ferritin as well as canine liver ferritin. To examine the specificity of canine IgM and IgA to ferritin H and L subunits, we used canine heart ferritin and canine liver ferritin with H/L subunit ratios of 3.69 and 0.43, respectively. Canine IgM and IgA recognized both of the H- and L-subunit-rich isoferritins, showing that their binding activities to ferritin depend on the H-subunit content. Recombinant bovine H-chain ferritin homopolymer expressed in a baculovirus expression system bound more with IgM and IgA than the recombinant L-chain homopolymer expressed under the same conditions. These results suggest that canine IgM and IgA recognize H-subunit-rich isoferritins, and that H-subunit-rich isoferritins are cleared from the circulation more rapidly than L-subunit-rich isoferritins.  相似文献   

5.
Three monoclonal antibodies against human liver ferritin were selected to study antigenic determinants (epitopes) of human isoferritins. These monoclonal antibodies were found to form immunoprecipitin lines with ferritin in double diffusion tests (Ouchterlony), indicating multiple epitopes on a single ferritin molecule. The antibodies revealed high species specificity as well. Monoclonal antibodies MA301 and MA311 appeared to recognize different epitopes, since they did not inhibit each other in competitive enzyme-linked immunosorbent assay (ELISA). However, MA309 recognized both epitopes for MA301 and MA311 with similar competitive inhibition. These epitopes were not detectable when ferritin was treated with 8M urea (pH 2.5) and were detectable upon reconstruction by dialysis against 2 M urea (pH 7.2), suggesting that these monoclonals recognize epitopes in the tertiary structure of the ferritin molecule. As a matter of fact, these monoclonals react preferentially with intact ferritin molecule and only negligibly with subunits. Isoelectric focusing patterns of human ferritins demonstrated that liver, spleen, placenta, and hepatoma cells (Li-7) transplanted in nude mice contained basic isoferritins, whereas HeLa cells (carcinoma), Wa cells (EB virus-transformed B cells), and Raji cells (Burkitt's lymphoma) contained acidic isoferritins. Human heart ferritin displayed a somewhat intermediate pattern between liver and HeLa ferritins. In spite of the heterogeneous population of human isoferritins, the dissociation constants (Kd) of the three monoclonal antibodies to liver, HeLa, and heart isoferritins were quite similar.  相似文献   

6.
Properties of human tissue isoferritins.   总被引:7,自引:1,他引:6       下载免费PDF全文
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.  相似文献   

7.
In rats with chronic dietary iron overload, a higher amount of liver ferritin L-subunit mRNA was found mainly engaged on polysomes, whereas in control rats ferritin L-subunit mRNA molecules were largely stored in ribonucleoprotein particles. On the other hand, ferritin H-subunit mRNA was unchanged by chronic iron load and remained in the inactive cytoplasmic pool. In agreement with previous reports, in rats acutely treated with parenteral iron, only the ferritin L-subunit mRNA increased in amount, whereas both ferritin subunit mRNAs shifted to polysomes. This may indicate that, whereas in acute iron overload the hepatocyte operates a translation shift of both ferritin mRNAs to confront rapidly the abrupt entry of iron into the cell, during chronic iron overload it responds to the slow iron influx by translating a greater amount of L-subunit mRNA to synthesize isoferritins more suitable for long-term iron storage.  相似文献   

8.
Abstract: The brain requires a ready supply of iron for normal neurological function, but free iron is toxic. Consequently, iron bioavailability must be stringently regulated. Recent evidence has suggested that the brain iron regulatory system is dysfunctional in neurological disorders such as Alzheimer's and Parkinson's diseases (AD and PD, respectively). A key component of the iron regulatory system in the brain is ferritin. Ferritin consists of 24 subunits, which are distinguished as either a heavy-chain (H) or light-chain (L) isoform. These peptide subunits are genetically and functionally distinct. Thus, the ability to investigate separately the types of ferritin in brain should provide insight into iron management at both the cellular and the molecular level. In this study, the ratio of isoferritins was determined in select regions of adult elderly AD and PD human brains. The H-rich ferritin was more abundant in the young brain, except in the globus pallidus where the ratio of H/L ferritin was 1:1. The balance of H/L isoferritins was influenced by age, brain region, and disease state. With normal aging, both H and L ferritin increased; however, the age-associated increase in isoferritins generally failed to occur in AD and PD brain tissue. The imbalance in H/L isoferritins was disease and region specific. For example, in frontal cortex, there was a dramatic (fivefold) increase in the ratio of H/L ferritin in AD brains but not in PD brains. In PD, caudate and putamen H/L ratios were higher than in AD and the elderly control group. The analysis of isoferritin expression in brain provides insight into regional iron regulation under normal conditions and suggests a loss of ability to maintain iron homeostasis in the two disease states. This latter observation provides further evidence of dysfunction of iron homeostatic mechanisms in AD and PD and may contribute significantly to understanding the underlying pathogenesis of each, particularly in relation to iron-induced oxidative damage.  相似文献   

9.
The six isoferritins in normal human liver and spleen with pIs between 5.7 and 5.2 were fractionated according to iron content by sucrose density gradient centrifugation. The pIs and subunit composition of the isoferritins in fractions from the gradients were determined by gel electrofocussing and electrophoresis in acid/urea polyacrylamide gels respectively. Fractions with the lowest iron content contained the two basic isoferritins, which were homopolymers of the L subunit and had pIs of 5.7 and 5.6. Increasing iron content correlated with decreasing isoelectric point and an increasing substitution of the H subunit in the isoferritin shell. The two isoferritins with highest iron content consisted of 92% and 8% respectively of the L and H subunits and had pIs of 5.3 and 5.2.  相似文献   

10.
The biochemical characteristics of human placental and hepatic ferritins were compared. By DEAE cellulose column chromatography, placental ferritin was found to contain more acidic isoferritins, while hepatic ferritin was richer in basic isoferritins. The electrofocusing patterns of subunits revealed that placental ferritin contained at least one unique subunit on the acidic side and might lack one subunit on the basic side as compared with hepatic ferritin. It is hypothesized that microheterogeneities, immunological as well as biochemical, existing between placental and hepatic ferritins cannot be explained merely in terms of different proportions of known acidic and basic subunits, but appear to have resulted from the existence of the unique acidic subunit of placental ferritin and possibly also a unique basic subunit of hepatic ferritin.  相似文献   

11.
On polyacrylamide slab electrophoresis two ferritin species, termed fast and slow, were present in three control and four iron-loaded human hearts. The ratio of the fast to slow species varied between hearts and correlated with the distribution of isoferritins, which had pI values ranging from 4.8 to 5.6. The acidic isoferritins were prominent in all hearts, but one control and the four iron-loaded hearts contained, in addition, basic isoferritins. All ferritins were composed of two subunits, an acidic H type and a more basic HL type, which was the main subunit in normal liver. Hearts with the highest proportions of acidic isoferritin contained the highest proportion of the H subunit.  相似文献   

12.
The role of iron-dependent oxidative metabolism in protecting the oxidable substrates contained in mature adipocytes is still unclear. Because differentiation increases ferritin formation in several cell types, thereby leading to an accumulation of H-rich isoferritins, we investigated whether differentiation affects iron metabolism in 3T3-L1 pre-adipocytes. To this aim, we evaluated the expression of the genes coding for the H and L ferritin subunits and for cytoplasmic iron regulatory protein (IRP) during the differentiation of 3T3-L1 cells in adipocytes induced by the addition of isobutylmethylxanthine, insulin, and dexamethasone. Differentiation enhanced ferritin formation and caused overexpression of the H subunit, thus altering the H/L subunit ratio. Northern blot analysis showed increased levels of H subunit mRNA. A gel retardation assay of cytoplasmic extract from differentiated cells, using an iron-responsive element as a probe, revealed enhanced an RNA binding capacity of IRP1, which correlated with the increase of IRP1 mRNA. The observed correlation between differentiation and iron metabolism in adipocytes suggests that an accumulation of H-rich isoferritin may limit the toxicity of iron in adipose tissue, thus exerting an antioxidant function.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
We have isolated essentially full-length cDNA clones for human ferritin H and L chains from a human liver cDNA library. This allows the first comparison of H and L nucleotide and amino acid sequences from the same species as well as ferritin L cDNA sequences from different species. We conclude that human H and L ferritins are related proteins which diverged about the time of evolution of birds and mammals. We also deduce the secondary structure of the H and L subunits and compare this with the known structure of horse spleen ferritin. We find that residues involved in subunit interaction in shell assembly are highly conserved in H and L sequences. However, we find several interesting differences in H subunits at the amino acid residues involved in iron transport and deposition. These substitutions could account for known differences in the uptake, storage, and release of iron from isoferritins of different subunit composition.  相似文献   

16.
Ferritin was isolated from porcine heart, liver and spleen. Reversed phase high performance liquid chromatography of the ferritin subunits yielded three chromatographic fractions. The relative proportions of the three chromatographic fractions were different for each tissue ferritin. These results support the model which proposes a combination of (at least) two subunit types as the basis for the existence of isoferritins.  相似文献   

17.
18.
Unfractionated pure ferritin isolated from the livers of female or male mice forms five narrow protein-positive bands on standard 5% polyacrylamide gel disc electrophoresis (pH 9.0). Since all of these sub-bands also contain iron, they are interpreted as being isoferritins. The multiple sub-bands are very unlikely to be artifactually generated by the analytical procedure used since they are not found in horse spleen ferritin when this is coelectrophoresed with female mouse liver ferritin. The present results provide an independnet indication that many isoferritins indeed can be found within a single organ.  相似文献   

19.
The iron storage protein ferritin was purified to electrophoretic homogeneity from cow uterine myometrium. Its Mr did not exceed 440, 000 and H-chains predominated in the subunit composition; the iron saturation was 43 iron ions per protein molecule. The uterine myometrial ferritin was a potent natural modulator of Ca-calmodulin-independent phosphodiesterase (Ca-CM-independent PDE, EC 3.1.4.17) isolated from the same tissue. Addition of iron-poor ferritin from uterine myometrium and iron-reach liver ferritin caused three- and two-fold inhibition of the enzyme activity, respectively. The iron transport protein transferrin in iron-saturated and iron-depleted forms can also inhibit Ca-CM-independent PDE activity by two-fold. In both cases, the degree of saturation with iron was not crucial for the inhibitory effects of these proteins on the enzyme activity. These data suggest that iron homeostasis proteins can modulate the cyclic nucleotide level in non-nervous tissue via interaction with enzymes involved in cyclic nucleotide hydrolysis.  相似文献   

20.
Previously we have demonstrated an apoptosis inducing activity for a rat hepatocyte conditioned medium (CM) presumably mediated by acidic isoferritins. Here, we present support for this assumption since isoferritins purified from different rat hepatocyte CM significantly enhanced the frequency of apoptotic cells in primary rat hepatocytes, an effect completely inhibited by a neutralizing anti-H-ferritin antibody. The apoptosis induction appears to be related to a 43 kDa ferritin subunit contained in the isoferritins released from primary hepatocytes, presumably representing a ferritin heavy/light chain heterodimer. In addition, these isoferritins immunologically crossreact with antibodies raised against placental isoferritin p43-PLF (which also contains a 43 kDa ferritin subunit) and melanoma-derived H-chain ferritin, representing ferritin isoforms which reveal immunomodulatory properties. Furthermore, p53 and FasL are upregulated upon isoferritin treatment in a time dependent mode, and apoptosis induction can be suppressed by neutralizing anti-FasL antibodies. Proapoptotic Bid is upregulated too and translocated into mitochondria in primary hepatocytes exposed to the isoferritins purified from the CM. Finally, epidermal growth factor (EGF) and dexamethasone (DEX), which counteract proapoptotic mitochondrial signalling, almost completely abolished the proapoptotic effect of the hepatocyte derived isoferritins. In conclusion, our findings demonstrate that acidic isoferritins with homology to immunomodulatory ferritin isoforms (p43-PLF, melanoma-derived-H-chain ferritin) are released from hepatocytes in vitro, and are able to stimulate upregulation of p53 and mediate apoptosis involving Fas (CD95) signalling as well as addressing the intrinsic mitochondrial proapoptotic pathway.  相似文献   

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