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PubMed1976

Hydrogen ion interactions of horse spleen ferritin and apoferritin.

The interactions of horse spleen ferritin and its derivative apoferritin with H+ ions were studied by potentiometric and spectrophotometric titration; to aid in data analysis, heats of ionization over a limited pH range and amide content were also determined. Per apoferritin subunit, all tyrosine and cysteine side chains, two of the nine lysine side chains and at least three of the six histidine side chains were found not to titrate; a preliminary but self-consistent analysis of the titration data is proposed. The titration curve of ferritin was identical with that of apoferritin in the pH range 5.5 to 3. In addition, under the conditions used, the reactivities of ferritin histidines to bromoacetate and of ferritin lysines to formaldehyde were identical with those in apoferritin. Above pH 8, a time-dependent titration of the ferritin core occurs which prevents comparison of the titration curves of the two proteins in this region. However, in the pH regions 5.5 to 7.5, two extra groups per subunit titrate reversibly in ferritin relative to apoferritin. Moreover, although the isoionic points of ferritin and apoferritin are identical in water, the isoionic point of ferritin is 0.5 pH unit lower than that of apoferritin in 0.16 to 1 M KCl. The different effects of KCl and NaCl on the two proteins indicate the presence of cation binding sites in ferritin that are absent in apoferritin and possibly also the presence of anion binding sites in apoferritin that are occupied in ferritin by anions of the core. The difference between the isoionic points of the two proteins in KCl has been interpreted to indicate the presence of approximately 2 phosphate residues per ferritin subunit which serve as cation binding sites and which are negatively charged at the isoionic point in KCl. These phosphates may also represent the additional residues that titrate in ferritin between pH 5.5 and 7.5, or may interact with positively charged residues on the inner surface of the ferritin shell, or both.

Animals↗

PubMed1975

Characterization and subunit analysis of ferritin isolated from normal and malignant human liver.

Ferritin was purified from normal, fetal, and malignant liver tissue. Ferritin purified from hepatoma tissue migrated slightly faster than normal human liver ferritin in polyacrylamide gel electrophoresis. Hepatoma and fetal liver ferritin contained an acidic components in gel and liquid isoelectric focusing not found in normal liver ferritin. We have called it a carcinofetal isoferritin. The subunit compositions of ferritins purified from human liver cell carcinoma and normal liver were then compared. Both ferritin consisted of a subunit species with an identical molecular weight of approximately 18,500. A single subunit of similar molecular weight was also demonstrable after dissociation of 8 M urea and by gel filtration in urea. Two subunits were demonstrable in normal liver ferritin by means of acrylamide electrophoresis in 8 M urea in acid pH. The same two subunits were also demonstrable in ferritin isolated from human liver cell carcinoma. However, a third subunit, intermediate in charge between the two normal liver subunits, was demonstrable in different amounts in ferritins from two hepatomas. Ferritins from normal and malignant livers were immunologically indistinguishable. The tumor-specific acidic isoferritin was isolated and antisera were prepared. The isolated acidic isoferritin was found to be immunologically identical to normal liver isoferritins. It is concluded that the multiple isoferritins of the human liver ferritin consist of two subunits, which are identical in molecular weight but which differ in net charge. Ferritin, isolated from two human liver carcinoma tissues, was composed of the same two subunits and a third unique subunit. Different amounts of these subunits may account for the several normal isoferritins and a unique tumor-specific acid isoferritin found in hepatoma.

Carcinoma, Hepatocellular↗

PubMed1975

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.

Amino Acids↗

PubMed1979

The kinetics of serum and tissue ferritins: relation to carbohydrate content.

Significant differences were observed in the rate of disappearance from plasma of ferritins purified from rat serum and from different organs. Ferritin from all sources including purified serum ferritin was rapidly removed from plasma by the liver. No difference in biological half-life was observed between apoferritin prepared by ultracentrifugation of liver ferritin and whole liver ferritin and iron-loaded animals cleared injected serum ferritin from plasma at a comparable rate to normal rats. When amounts of 100 microgram of ferritin were injected into rats the half-life was significantly lengthened. The study confirmed the fact that ferritin iron and ferritin protein were removed from plasma at the same rate. No consistent effect of acidic or more basic isoferritin composition on biological half-life was apparent. After chromatography on concanavalin A-Sepharose 6B those ferritins which were predominantly bound to Con A-Sepharose had a half-life which was approximately twice that of ferritins which did not bind. It is concluded that the variation in plasma disappearance of ferritins of different tissue origin was explainable on the basis of carbohydrate content of the molecule.

Animals↗

PubMed1979

Mouse hepatoma and liver ferritins. Comparative structural studies.

Pure ferritin from male mouse liver produces a single band of monomers (RF = 0.199) with electrophoresis in polyacrylamide gels at pH 9.0. The five sub-bands within this monomeric band appear to represent charge isomers having the same molecular size. Ferritin from BH3 transplantable mouse hepatoma shows two overlapping bands of monomers (RFA = 0.208 and RFB = 0.240); further electrophoretic studies show that these bands represent two subpopulations of molecules differing both in charge and size. Sub-bands are not found in this hepatoma ferritin. The larger tumor ferritin reaches the same end migration position as all liver isoferritins on gradient gels, signifying a very similar or identical molecular size; however, the absence of sub-bands indicates that this hepatoma ferritin differs in charge from the homologous liver proteins. Liver and hepatoma ferritins both produce a single prominent subunit band corresponding to nominal molecular weights of 22 250 and 21 700, with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. With electrophoresis on polyacrylamide gradient slabs containing sodium dodecyl sulfate and dithiothreitol, both liver and hepatoma ferritins now reveal two subunits bands situated at identical positions. The polypeptides of these two closely spaced bands have a nominal molecular weight difference of less than 1000. Neither the hepatoma nor the liver seems to produce the ferritins found in the other tissue. Nevertheless, all these ferritins are composed of the same two types of subunits, albeit in different relative amounts. Observed distinctions in the ferritins from these normal or neoplastic cells must reflect differences in assembly and processing, as well as in the regulated expression of the same ferritin genes.

Animals↗

PubMed1979

Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431.

We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4 degrees C and processed for transmission electron microscopy. Under these conditions, approximately 6 X 10(5) molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37 degrees C, cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37 degrees C, 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing 125I-EGF and fluorescein-conjugated EGF.

Carcinoma, Squamous Cell↗

PubMed1978

Variable effects of iron status on the concentration of ferritin in rat plasma, liver, and spleen.

The present studies were conducted to determine the relationships between iron status and ferritin levels in plasma, liver, and spleen of rats. Rats were fed either iron-adequate or iron-deficient purified diets, and measurements of hemoglobin and plasma and tissue ferritin levels were made at various times during iron depletion and iron repletion. Although mean plasma ferritin concentrations of iron-deficient rats were directionally less than those of iron-adequate rats, these differences were not statistically significant due to high variability among similarly treated animals. During iron repletion plasma ferritin concentrations again were so variable that no significant effect of iron repletion on plasma ferritin concentrations was observed. On the other hand, liver and spleen ferritin concentrations of similarly treated rats were much less variable. Ferritin liver and spleen stores decreased more rapidly than hemoglobin during iron deficiency and were restored more slowly than hemoglobin during iron repletion. There was no evidence of correlation between liver and plasma ferritin concentration. Because of the variable responses of plasma ferritin concentration to iron depletion and repletion and the lack of relationship between plasma and liver ferritin concentrations, it is concluded that plasma ferritin concentration is not a good indicator of iron status in rats.

Animals↗

PubMed1979

Suppressive effect of ferritin on in vitro lymphocyte function.

This study describes the effect of ferritin on lymphocyte function in vitro. Peripheral blood lymphocytes isolated from normal donors were incubated with purified human splenic ferritin, and the mitogenic effect of phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and mixed lymphocyte reaction (MLR) were assessed by the uptake of 3H-thymidine (3H-TdR). Ferritin (0.25--5.0 micrograms/ml culture) caused a marked suppression of PHA nad Con A blastogenesis but had no suppressive effect on PWM-induced transformation. Maximal suppression was obtained at a ferritin concentration of 1 microgram/ml and this was not enhanced by increasing ferritin concentrations. Ferritin also reduced the Con A capping phenomenon in normal lymphocytes from 22% to 6%, suppressed the MLR reaction but had no effect on the ability of normal lymphocytes to form E, EA and EAC rosettes or on in vitro lymphocyte cytoxicity against the K-562 cell line. Visual proof of the suppressive effect of ferritin on mitogen induced blastogenesis was provided by scanning electron microscopy, and direct evidence for the ability of lymphocytes to bind ferritin was obtained from studies with radioiodine labelled ferritin. The above findings indicate that ferritin suppresses certain parameters of T-lymphocyte function in vitro. The relation of the present findings to recognized abnormalities of T-cell function encountered in certain neoplastic disorders associated with high serum ferritin levels is at present unknown.

Cytotoxicity, Immunologic↗

PubMed1978

Radioimmunoassay of serum ferritin.

Purified human spleen ferritin was labelled with 125I. On Sepharose 6-B gel filtration four species of labelled products were separated: a component with a higher molecular weight than ferritin; a component which is eluted in the same volume as unlabelled ferritin; and two labelled compounds with molecular weights lower than ferritin. When these labelled materials were used in a double antibody radioimmunoassay, the high molecular weight fraction showed variable and high non-specific binding and was poorly displaced by unlabelled ferritin; the fraction behaving like true ferritin gave good standard curves and showed non-specific binding of less than 1%. The remaining two components showed poor binding to rabbit antiferritin. Using labelled material from the second fraction, a double antibody radioimmunoassay capable of measuring 2 microgram ferritin protein/litre of serum was developed. Inter- and intra-assay variation was between 3% and 8% over a concentration range of 0 to 250 microgram ferritin protein/litre. Good agreement between serum ferritin levels assayed by the present method and by an immunoradiometric method was obtained. Labelled ferritin was stable for at least six weeks. The simplicity of the methodology makes it possible to assay serum ferritin in large batches.

Ferritins↗

PubMed1975

Distribution of ferritin-conjugated lectins on sialidase-treated membranes of human erythrocytes.

The labeling of sialidase-treated, human erythrocyte membranes with ferritin-conjugates of four plant lectins, concanavalin A, Ricinus communis hemagglutinin, Bauhinia purpurea hemagglutinin and Arachis hypogoea hemagglutinin, is reported. Among these ferritin-conjugated lectins, ferritin-conjugated concanavalin A and ferritin-conjugated R. communis hemagglutinin were found in clusters on the sialidase-treated membranes, whereas ferritin-conjugated B. purpurea hemagglutinin and ferritin-conjugated A. hypogoea hemagglutinin were found in a random distribution on the membranes. Furthermore, when the membranes were labeled with a mixutre of concanavalin A and ferritin-conjugated B. purpurea hemagglutinin, ferritin particles were found in clusters, indicating that the membrane receptors for B. purpurea hemagglutinin were forced to more together with those for concanavalin A. A method for the quantitative estimation of the clustering of ferritin particles on the membranes was also devised and applied to the labeling of sialidase-treated, human erythrocyte membranes with the above four ferritin-conjugated lectins.

Binding Sites↗

PubMed1976

The purification and properties of ferritin from human serum.

1. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70 degrees C. After removal of denatured protein, ferritin was concentrated and further purified by ion-exchange chromatography and gel filtration. In most cases, only a partial purification was achieved. 3. In method B, ferritin was extracted from the serum with a column of immuno-adsorbent [anti-(human ferritin)] and released from the column with 3M-KSCN. Further purification was achieved by anion-exchange chromatography followed by the removal of remaining contaminating serum proteins by means of a second immunoadsorbent. Purifications of up to 31 000-fold were achieved, and the homogeneity of the final preparations was demonstrated by polyacrylamide-gel electrophoresis. 4. Serum ferritin purified by either method has the same elution volume as human spleen ferritin on gel filtration on Sephadex G-200. Serum ferritin has a relatively low iron content and iron/protein ratios of 0.023 and 0.067 (mug of Fe/mug of protein) were found in two pure preparations. On anion-exchange chromatography serum ferritin has a low affinity for the column when compared with various tissue ferritins. Isoelectric focusing has demonstrated the presence of a high proportion of isoferritins of relatively high pI. 5. Possible mechanisms for the release of ferritin into the circulation are briefly discussed.

Chromatography, Ion Exchange↗

PubMed1978

Dynamics of toxin and lectin receptors on a lymphoma cell line and its toxin-resistant variant using ferritin-conjugated, 125I-labeled ligand.

The dynamics of the toxin Ricinus communis agglutinin II (RCAII or ricin) on cells of a murine lymphoma line (BW5147) and a toxin-resistant variant line (BW5147RicR.3) that is 200 times more resistant than the parent to direct RCAII cytotoxicity were examined using ferritin-conjugated, affinity purified, 125I-labeled RCAII (ferritin-125I-RCAII). Ferritin-125I-RCAII was indistinguishable from native RCAII in quantitative binding and cytotoxicity experiments. When RCAII-sensitive BW5147 and -resistant BW5147RicR.3 cells were labeled with ferritin-125I-RCAII at various toxin concentrations (1--10 microgram/ml), no differences in toxin binding were observed. These same cells were examined by electron microscopy. At low ferritin-125I-RCAII concentrations (1-3 microgram/ml RCAII) where only the parental BW5147 cells were significantly more sensitive to RCAII, toxin receptors were internalized by ferritin-125I-RCAII-induced endocytosis. In parallel experiments, ferritin-125I-RCAII that bound to the resistant BW5147RicR.3 cells remained relatively dispersed or clustered, and there was little evidence of transport into cells via endocytosis. At higher ferritin-125I-RCAII concentrations (greater than 7 microgram/ml RCAII) where both parental and resistant variant cells are sensitive to the cytotoxic effects of RCAII, more ferritin-conjugated toxin was bound, and subsequent endocytosis occurred to a similar degree in both cell types. Endocytosis of ferritin-conjugated concanavalin A was indistinguishable on RCAII-sensitive parental and resistant variant cells at all concentrations tested. The results suggest that a specific defect on the selected BW5147RicR.3 cells prevents RCAII entry into these cells a low toxin concentrations, rendering them more resistant to the cytotoxic effects of RCAII.

Animals↗

PubMed1979

The labelling of vesicles in frog endothelial cells with ferritin.

1. The labelling of endothelial cell vesicles with ferritin has been investigated by electron microscopy. Single capillaries in the frog mesentery have been perfused with solutions of known concentrations of ferritin for known periods before fixing the tissue in situ by superfusion with osmium tetroxide. 2. At 17 degrees C, the percentage of lumenal vesicles labelled with ferritin increased as the period of perfusion was increased up to 16 sec prior to fixation. When perfusions were longer than 16 sec, the percentage of vesicles labelled with ferritin remained fairly constant at 70%. 3. At 3 degrees C, no more than 25% of the lumenal vesicles were labelled during the first 30 sec. 4. After correcting the data for losses of ferritin due to sectioning, the distribution of ferritin molecules in the lumenal vesicles was consistent with a Poisson distribution. 5. After perfusions of 16 sec or longer, the number of ferritin molecules per labelled vesicle was roughly three to four times less than would be predicted from the lumenal concentration. 6. At all times there was a gradient of vesicles labelled with ferritin across the endothelial cells, i.e. the percentage of lumenal vesicles labelled was greater than that for cytoplasmic vesicles which in turn was greater than that for vesicles at the ablumenal surface. 7. Whereas the labelling of lumenal vesicles from zero time up to 16-20 sec, the main increase in labelling of cytoplasmic vesicles occurred between 10 and 20 sec. 8. It is concluded that there is a major diffusion barrier to ferritin molecules either close to the endothelial cell surface or across the necks of the lumenal vesicles. It also appears that ferritin molecules do not have access to vesicles during the latter part of their residence at the lumenal surface.

Animals↗

PubMed1979

Ferritin in rat kidneys with specific lesions due to a single dose of lead.

In the proximal tubular cells of rats or mice given a single, parenteral dose of lead, clusters of ferritin are frequently associated with characteristic cytoplasmic fibrillar bodies. To learn more about this relationship, we have investigated content and synthesis of ferritin protein and incorporation of iron into ferritin in rat kidneys 48 hours after a single parenteral dose of lead (10 microgram/g). By immunoradiometric assays, we found that the kidneys of female rats, whether treated with lead or not, contained significantly more ferritin protein than did kidneys of males of the same age and provenance. Administration of lead diminished (or did not significantly alter) the incorporation of 14C-amino acids into newly synthesized ferritin protein. Contrary to expectation, administration of lead tended to depress incorporation of 59Fe into kidney ferritin in rats maintained on standard rations and distilled water. Electron microscopy confirmed the presence of clusters of ferritin in close association with dense fibrillar bodies in the cytoplasm of proximal tubular cells of rats given lead. Considered together, the findings indicate that clustering of ferritin next to the dense fibrillar cytoplasmic lesions is a selective effect of lead that requires neither augmented synthesis of ferritin protein nor increased incorporation of iron into preexisting ferritin.

Amino Acids↗

PubMed1977

Structural features of rat cardiac ferritins.

Ferritin extracted from rat heart containes two species separable by gel electrophoresis. These were purified and examined for structural characteristics. As in gel electrophoresis, cardiac ferritin preparations yielded only two bands on isoelectric focusing in gels, with pI values of 4.6 and 4.8. After separation by preparative electrophoresis, the two species were found to have a different amino acid composition from each another and from liver ferritin. Similarly, peptide maps showed several components not found in liver ferritin. On dissociation and electrophoresis with sodium dodecyl sulfate, heart ferritins were found to contain subunits of the same sizes as in other rat ferritins but also some larger components. Since cardiac ferritins have apparent molecular weights greater than those of other ferritins, it is concluded they probably contain more subunits, and possibly some of larger size not present in ferritins of other tissues.

Amino Acids↗

PubMed1979

Alteration in tryptic peptide patterns of ferritins purified from human colon carcinoma.

Ferritin from malignant tissue differs electrophoretically from normal ferritin. The molecular basis of this difference has not yet been defined. Malignant tissue contains a mixture of ferritins from normal cells, inflammatory cells as well as cancer cells. GW-39 is a pure colon carcinoma cell system that synthesizes human carcinoembryonic antigen. Therefore, ferritin was isolated from normal colon mucosa and colon cancer tissues, as well as from the colon carcinoma cell line, to clarify the molecular relationship between normal and malignant ferritins. Colon carcinoma ferritin differs in primary structure from normal colon mucosal ferritin and contains at least six additional different tryptic peptides. These six peptides were also found in the ferritin from the colon carcinoma cell line. These data suggest that the alteration in ferritin structure occurs at the cellular level and is associated with the malignant state.

Amino Acids↗

PubMed1979

Characterization and localization of human placental ferritin.

Ferritin has been purified from normal full-term human placentae and its antigenic and molecular characteristics compared with adult liver ferritin. Placental ferritin is composed predominantly of a single subunit type, co-migrating with a liver ferritin standard on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Comparison of dose-response curves in an immunoradiometric assay indicated some tissue-specific antigenicity for placental ferritin. This was supported by immunofluorescence studies on cryostat sections of human placentae by using antibodies to placental and spleen ferritin. Specific staining for placental ferritin was demonstrated within placental syncytiotrophoblast, particularly localized towards the microvillus plasma membrane. Ferritin has also been shown by electrophoretic and antigenic analysis to be present in protein fractions solubilized from isolated human syncytiotrophoblast microvillus plasma-membrane preparations, suggesting that ferritin may play an active role in the transfer of iron from maternal transferrin across the syncytiotrophoblast plasma membrane.

Chemical Phenomena↗

PubMed1979

Binding of human serum ferritin to concanavalin A.

1. A high proportion of the ferritin in normal serum binds to concanavalin A. Binding is prevented by the addition of alpha-D-methylglucoside to the reaction mixture. 2. Ferritin in extracts of normal heart, liver and spleen or serum ferritin from patients with massive hepatic necrosis does not bind to concanavalin A. 3. Isoelectric focusing of preparations of serum ferritin from patients with primary haemochromatosis shows that the ferritin fraction binding to concanavalin A consists, predominantly, of the more acidic isoferritins. 4. These findings suggest that carbohydrate residues may be added to ferritin during its secretion into the plasma. Glycosylation may account for the heterogeneity of serum ferritin on isoelectric focusing. 5. Direct release of intracellular ferritin from damaged tissue may be indicated by an increase in the proportion of circulating ferritin which does not bind to concanavalin A. Such an increase has been found in sera from patients with iron overload.

Concanavalin A↗