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At least 19 recordsLinked to original sources
PubMed1976

Chemical modification as a probe of the topography and reactivity of horse-spleen apoferritin.

In apoferritin, but not in ferritin, 1.0 +/- 0.1 cysteine residue per subunit can be modified. In ferritin 3.3 +/- 0.3 lysine residues and 7.1 +/- 0.7 carboxyl groups per subunit can be modified, whilst the corresponding values for apoferritin are 4.4 +/- 0.4 lysine residues and 11.0 +/- 0.4 carboxyl groups per subunit. Modification of lysine residues which maleic anhydride and carboxyl groups with glycineamide in apoferritin which has been dissociated and denatured in guanidine hydrochloride leads to the introduction of 9.1 +/- 0.5 maleyl groups per subunit and 22.0 +/- 0.9 glycineamide residues per subunit. Whereas unmodified apoferritin subunit can be reassociated from guanidine hydrochloride to apoferritin monomer, the ability of maleylated apoferritin to reassociate is impaired. Apoferritin in which all the carboxyl groups have been blocked with glycineamide cannot be reassociated to apoferritin and exists in solution as stable subunits. The modification of one cysteine residue per subunit, of 3 or 4 lysine residues per subunit or of 7 carboxyl groups per subunit has no effect on the catalytic activity of apoferritin. In contrast the modification of 11 carboxyl groups per subunit completely abolishes the catalytic properties of the protein. We conclude that one or more carboxyl groups are essential for the catalytic activity of horse spleen apoferritin.

Amides↗
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↗
PubMed1976

Comparison of the physical properties of chemically prepared and tissue-endogenous equine apoferritins.

To support the case for a biological applicability of previous experiments performed on chemically prepared apoferritin, its hydrodynamic and circular dichroic properties were compared to those of tissue-endogenous apoferritin. The molecular weights and sedimentation coefficients of the two differently prepared apoferritins were identical. Similarly, their circular dichroic spectra between 310 and 200 nm were identical. These data support the hypothesis that chemically prepared apoferritin is identical with tissue-endogenous apoferritin. Ferritins exhibited low ultraviolet CD spectra quite similar to apoferritin except amplitudes were often as much as 8% less. Apoferritin subunits, prepared by acid dissociation, did not possess the positive 292 and 286 nm bands exhibited by the native molecule, although hydrodynamically the subunit behaves as a globular protein. This suggests the presence of tryptophanyl and tyrosyl residues at the subunit contact sites. Two characteristics of the apoferritin CD spectrum were proposed as means to evaluate the quality of apoferritin preparations. These are a ratio, [theta]286 [theta]222 = -4.5 X 10(-3) +/- 0.2 X 10(-3), and the wavelength region, 258 to 262 nm, at which the near ultraviolet CD spectrum goes from a positive to a negative sense.

Animals↗
PubMed1976

Studies on iron uptake and micelle formation in ferritin and apoferritin.

Iron uptake and micelle formation in ferritin and apoferritin have been followed both spectrophotometrically and by means of sedimentation velocity experiments. Information was thus obtained on the molecular weight distribution of the reconstitution product. To achieve incorporation 'native' ferritin (whole ferritin as purified from horse spleen), 'native' apoferritin (apoferritin prepared by fractionation of ferritin preparations) and 'reduced' apoferritin (apoferritin prepared by reduction of ferritin by dithionite or ascorbic acid) have been incubated with ferrous salts in the presence of oxidizing agents under different experimental conditions. Although some iron is incorporated in 'native' ferritin, full saturation is not achieved and the molecular weight distribution of the incubated products remains heterogeneous. 'Native' and 'reduced' apoferritin show a similar iron incorporation, but the reconstitution products markedly differ in terms of their iron distribution. Ferritin reconstituted from 'native' apoferritin has a broad molecular weight distribution, while that reconstituted from 'reduced' apoferritin is characterized by a narrow, homogeneous molecular weight distribution. However treatment of apoferrition with reducing or oxidizing agents prior to the incubation alters the characteristics of the iron distribution without changing the iron incorporation properties. These results point to a role of the protein moiety not only in iron oxidation, but also in micelle formation.

Animals↗
PubMed1992

In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.

Amino Acids↗
PubMed1975

In vitro stimulation of apoferritin synthesis by iron.

The apparent induction of apoferritin synthesis by iron has been examined in cell-free systems from rat and rabbit liver. Both systems allowed the complete synthesis de novo of apoferritin isolated by chromatographic or immunological means. Addition of iron at levels of 0.2--1 mM specifically stimulated incorporation of radioactive amino acids into apoferritin purified after classical heat extraction. The effect was also observed when iron was added at the end of the incubation period in the absence of continuing protein synthesis. Further, iron addition had no effect on the amount of newly synthesised apoferritin subunits as estimated by direct immunological precipitation from the reaction mixture. These results suggest that iron acts at some stage subsequent to translation in stimulating apoferritin biosynthesis.

Animals↗
PubMed1978

Primary structure of rat liver apoferritin. The amino end.

Rat liver apoferritin is known to have a blocked amino end. From a pronase digest of rat liver apoferritin we have isolated and purified by ion-exchange chromatography the blocked N-terminal tripeptide. Its sequence and the nature of the blocking group were shown to be Ser-Ser-Gln and an acetyl moiety, respectively. The N-terminal sequence of rat liver apoferritin is thus N-acetyl-Ser-Ser-Gln, which coincides with the N-terminal sequence of horse-spleen apoferritin, the only other apoferritin studied structurally at present.

Amino Acid Sequence↗
PubMed1977

The properties of Corbicula sandai apoferritin.

Corbicula sandai apoferritin possesses physical properties different from apoferritins of other species. The native molecular weight was estimated from its s020,w of 18.7 S to be about 503 000. Empirical molecular weight estimation methods in denaturing solvents yielded a molecular weight estimate for the constituent polypeptide chain of 23 000. The circular dichroic spectrum of C. sandai apoferritin was significantly different from other apoferritins and it was immunologically unreactive with rabbit anti-human ferritin antisera.

Amino Acids↗
PubMed1979

Ferritin- and apoferritin-induced immune complex glomerulonephritis in mice.

In order to study the effects of the protein moiety independent of the protein-iron complex in the development of ferritin-induced glomerulonephritis, we compared the effects of ferritin, equimolar amounts of apoferritin, and equimolar amounts of iron dextran in Swiss albino mice. The results were compared to both saline-injected and non-injected controls. Ferritin resulted in a glomerulonephritis associated with predominantly mesangial deposition of immune complexes. Tubulo-interstitial changes occurred as well. Iron dextran resulted in similar but less severe tubulo-interstitial changes and evoked no glomerular alterations. Apoferritin resulted in an immune complex glomerulonephritis usually associated with membranous deposits. No tubular or interstitial changes occurred. Proteinuria developed in animals receiving apoferritin. Since the protein-iron complex caused tubular and interstitial damage, apoferritin may provide a more suitable model of immune-complex-mediated glomerulonephritis.

Animals↗
PubMed1976

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

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

Animals↗
PubMed1992

Uranium-loaded apoferritin with antibodies attached: molecular design for uranium neutron-capture therapy.

A method is described to deliver 235U to tumors; the isotope would then be fissioned by incident neutrons, producing localized lethal radiation sufficient for therapy. Apoferritin was loaded with an average of approximately 800 238U atoms per molecule. Stability of the loaded apoferritin in solution was improved, so that only 8% loss of uranium occurred after 8 days at pH 7. Fab' antibody fragments were covalently attached to the uranium-loaded apoferritin, and the immunoreactivity of the conjugate was 92% of that for antibody alone. Such bio-uranium constructions should provide significant advantages over boronated antibodies to meet the requirements for clinical neutron-capture therapy.

Apoferritins↗
PubMed1976

Synthesis of apoferritin in mouse peritoneal macrophages. Characterization of 20 S particles.

Apoferritin particles were found in mouse peritoneal macrophages cultured in vitro. They were found as 20S particles in the "ribosomal fraction" of macrophages labeled with L-[14C]glutamic acid. Possibilities that they were breakdown products of ribosomes or of other well-known contaminants of the ribosomal fraction were excluded because they did not incorporate [5-3H]uridine. They were resistant to RNase and were relatively resistant to detergent. The antibody against horse spleen apoferritin precipitated about 70% of the particles in the 20S region, judging by measurement of radioactivity. On in vitro incubation with Fe2+ and suitable oxidizing agents the sedimentation coefficient of 80% of the 20S particles changed to about 60S, which corresponds to that of ferritin. SDS-polyacrylamide gel electrophoresis revealed the presence of subunit structures with the same molecular size as that of mouse liver apoferritin. Under the electron microscope, the particles appeared spherical with a relatively uniform diameter of about 130 A.

Animals↗
PubMed1977

Characterization of serum ferritin in iron overload: possible identity to natural apoferritin.

Serum ferritins from a patient with haemochromatosis and from a patient with transfusional siderosis were compared with tissue isoferritins on the basis of their iron content, isoferritin spectrum and immunological properties. Both serum ferritins had a low iron content and corresponded to only the most basic isoferritins in liver. The serum ferritins were very similar to the natural apoferritin from liver in all respects.

Apoferritins↗
PubMed1992

The protein of M(r) 21,000 constituting the prosome-like particle of duck erythroblasts is homologous to apoferritin.

In duck erythroblasts, two major populations of untranslated messenger (m) RNP can be separated by sucrose gradient centrifugation in low ionic strength. One of these contains globin mRNA associated to protein factors, among them the prosomes. The other, sedimenting in the 35S zone, contains non-globin mRNA. From this '35S' mRNP, a new RNP particle called the prosome-like particle was isolated and characterized [Akhayat, O., Infante, A. A., Infante, D., Martins de Sa, C., Grossi de Sa, M.-F. & Scherrer, K. (1987) Eur. J. Biochem. 170, 23-33]. The PLP is a multimer of a protein of M(r) 21,000, and contains small RNA species. The particle is tightly associated with repressed mRNA and inhibits in vitro protein synthesis. We show here that the protein of M(r) 21,000, constituting the prosome-like particle, is apoferritin. Different approaches confirm the RNP character of this particle and provide evidence that some of its RNA species are tRNA. The hypothesis is discussed as to whether (apo-)ferritin might serve other functions in addition to iron storage.

Amino Acid Sequence↗
PubMed1979

Immunoradiometric and electroimmuno assay of increased ferritin and apoferritin levels in serum.

Ferritin in serum from patients with increased serum ferritin levels has been studied both quantitatively and qualitatively. All techniques utilized in these studies are suitable to be used as routine screening tests for large numbers of patients. Electroimmuno assay (EIA) has been compared with the solid phase immunoradiometric (IRMA) assay as a technique to determine serum ferritin concentration (r = 0.99) and is suggested as a useful alternative when determining ferritin concentrations above 500 microgram/l. Iron stained EIA gels have been used to indicate the iron content of the ferritin molecule in sera. This simple screening test has demonstrated that apoferritin is found more often than iron-rich ferritin in the serum of patients with elevated serum ferritin levels. Immunoelectrophoresis precipitin bands suggest the heterogeneity of ferritin in serum from different patients.

Animals↗