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A Treffry

Publications and source records attributed to A Treffry.

At least 37 records · Page 2Linked to original sources
PubMed1991

Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts.

Ferritin is important in iron homeostasis. Its twenty-four chains of two types, H and L, assemble as a hollow shell providing an iron-storage cavity. Ferritin molecules in cells containing high levels of iron tend to be rich in L chains, and may have a long-term storage function, whereas H-rich ferritins are more active in iron metabolism. The molecular basis for the greater activity of H-rich ferritins has until now been obscure, largely because the structure of H-chain ferritin has remained unknown owing to the difficulties in obtaining crystals ordered enough for X-ray crystallographic analysis. Here we report the three-dimensional structure of a human ferritin H-chain homopolymer. By genetically engineering a change in the sequence of the intermolecular contact region, we obtained crystals isomorphous with the homologous rat L ferritin and of high enough quality for X-ray diffraction analysis. The X-ray structure of human H ferritin shows a novel metal site embedded within each of its four-helix bundles and we suggest that ferroxidase activity associated with this site accounts for its rapid uptake of iron.

Amino Acid Sequence↗
PubMed1989

Identification of the ferroxidase centre in ferritin.

Ferroxidase activity in human H-chain ferritin has been studied with the aid of site-directed mutagenesis. A site discovered by X-ray crystallography has now been identified as the ferroxidase centre. This centre is present only in H-chains and is located within the four-helix bundle of the chain fold.

Animals↗
PubMed1989

Mössbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated Fe(III) atoms and oxo-bridged Fe(III) dimers as early intermediates.

Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room temperature before freezing). The measurements made at 4.1 and 90 K showed that for samples with n less than or equal to 40 at pH greater than or equal to 6.25 all iron was trivalent at tf = 3 min. Four different Fe(III) species were identified: solitary Fe(III) atoms giving relaxation spectra, which can be identified with the species observed before by EPR and UV difference spectroscopy; oxo-bridged dimers giving doublet spectra with large splitting, observed for the first time in ferritin; small Fe(III) clusters giving doublets of smaller splitting and larger antiferromagnetically coupled Fe(III) clusters, similar to those found previously in larger ferritin iron cores, which, for samples with n greater than or equal to 40, gave magnetically split spectra at 4.1 K. Both solitary Fe(III) and dimers diminished with time, suggesting that they are intermediates in the formation of the iron core. Two kinds of divalent iron were distinguished for n = 480, which may correspond to bound and free Fe(II).

Animals↗
PubMed1989

Recombinant H-chain ferritins: effects of changes in the 3-fold channels.

Human H-chain ferritins bearing sequence changes in the 3-fold channels have been expressed in E. coli to investigate the role of these channels in iron-storage processes. The proteins assemble into shells resembling those of native ferritins. Iron uptake measurements indicate that residues in the 3-fold channels are involved neither in initial Fe(II)-oxidation nor in iron-core nucleation.

Amino Acid Sequence↗
PubMed1989

Ferritin subunits in livers of siderotic mice.

The major ferritin species of mouse liver has been resolved by SDS-PAGE into two bands similar to the H and L subunits of rat liver ferritin with the L subunit predominating. Amino acid sequencing has confirmed the major, faster-migrating component as L chain. An additional, electrophoretically fast, minor ferritin was isolated from siderosome-containing subcellular fractions. In denaturing gels it gave a single 'F' subunit band of about 17 kDa, significantly smaller than the L and H subunits (about 20 and 21 kDa respectively). A small fragment isolated from the fast ferritin was sequenced. It corresponds to a 19-residue C-terminal peptide cleaved from L subunits in the assembled molecules. The F subunit must be derived from L subunits by loss of this peptide, and is not the expression product of a different gene. 'Fast' ferritins of siderotic mice and rats are thus analogous.

Amino Acid Sequence↗
PubMed1989

Release of iron from ferritin molecules and their iron-cores by 3-hydroxypyridinone chelators in vitro.

Ferritin molecules contain 24 subunits forming a shell around an inorganic iron-core. Release of iron(III) from ferritin and its isolated iron-cores by a series of hydroxypyridinone chelators with high affinities for iron(III) has been compared. The results collectively suggest that the chelators act by penetrating the protein shell and interacting directly with the iron-core in ferritin. Iron(III) is probably removed bound to a single ligand, but once outside the protein shell, the trihydroxypyridinone iron(III) complex predominates. The order of effectiveness of a group of pyridinones found for iron removal from ferritin molecules in solution differs from that obtained with hepatocytes in culture or with whole animals, where membrane solubility and other factors may modulate the response.

Animals↗
PubMed1988

Studies on haemosiderin and ferritin from iron-loaded rat liver.

Haemosiderin has been isolated from siderosomes and ferritin from the cytosol of livers of rats iron-loaded by intraperitoneal injections of iron-dextran. Siderosomal haermosiderin, like ferritin, was shown by electron diffraction to contain iron mainly in the form of small particles of ferrihydrite (5Fe2O3.9H2O), with average particle diameter of 5.36 +/- 1.31 nm (SD), less than that of ferritin iron-cores (6.14 +/- 1.18 nm). Mössbauer spectra of both iron-storage complexes are also similar, except that the blocking temperature, TB, for haemosiderin (23 K) is lower than that of ferritin (35 K). These values are consistent with their differences in particle volumes assuming identical magnetic anisotropy constants. Measurements of P/Fe ratios by electron probe microanalysis showed the presence of phosphorus in rat liver haemosiderin, but much of it was lost on extensive dialysis. The presence of peptides reacting with anti-ferritin antisera and the similarities in the structures of their iron components are consistent with the view that rat liver haemosiderin arises by degradation of ferritin polypeptides, but its peptide pattern is different from that found in human beta-thalassaemia haemosiderin. The blocking temperature, 35 K, for rat liver ferritin is near to that reported, 40 K, for human beta-thalassaemia spleen ferritin. However, the haemosiderin isolated from this tissue, in contrast to that from rat liver, had a TB higher than that of ferritin. The iron availability of haemosiderins from rat liver and human beta-thalassaemic spleen to a hydroxypyridinone chelator also differed. That from rat liver was equal to or greater, and that from human spleen was markedly less, than the iron availability from either of the associated ferritins, which were equivalent. The differences in properties of the two types of haemosiderin may reflect their origins from primary or secondary iron overload and differences in the duration of the overload.

Animals↗
PubMed1987

Reconstituted and native iron-cores of bacterioferritin and ferritin.

The structural and magnetic properties of the iron-cores of reconstituted horse spleen ferritin and Azotobacter vinelandii bacterioferritin have been investigated by high-resolution transmission electron microscopy, electron diffraction and Mossbauer spectroscopy. The structural properties of native horse spleen ferritin, native Az. vinelandii, and native and reconstituted Pseudomonas aeruginosa bacterioferritins have also been determined. Reconstitution in the absence of inorganic phosphate at pH 7.0 showed sigmoidal behaviour in each protein but was approximately 30% faster in initial rate for the Az. vinelandii protein when compared with horse spleen apoferritin. The presence of Zn2+ reduced the initial rate of Fe(II) oxidation in Az. vinelandii to 22% of the control rate. The iron-cores of the reconstituted bacterioferritins adopt defect ferrihydrite structures and are more highly ordered than their native counterparts, which are both amorphous. However, the blocking temperature for reconstituted Az. vinelandii (22.2 K) is almost identical to that for the native protein (20 K). Particle size measurements indicate that the reconstituted Az. vinelandii cores are smaller in median diameter than the native cores and this reduction in particle volume (V) offsets the increased magnetocrystalline contribution to the magnetic anisotropy constant (K) in such a way that the magnetic anisotropy barrier (KV), and hence the blocking temperature, is similar for both proteins. Reconstituted horse spleen ferritin exhibits a similar blocking temperature (38 K) to that determined for the native protein, although it is structurally more disordered. The possibility of introducing structural and compositional modifications in both horse ferritin and bacterioferritins by in-vitro reconstitution suggests that these proteins do not function primarily as a crystallochemical-specific interface for core development in vivo.

Animals↗
PubMed1987

Siderosomal ferritin. The missing link between ferritin and haemosiderin?

A minor electrophoretically fast component was found in ferritin from iron-loaded rat liver in addition to a major electrophoretically slow ferritin similar to that observed in control rats. The electrophoretically fast ferritin showed immunological identity with the slow component, but on electrophoresis in SDS it gave a peptide of 17.3 kDa, in contrast with the electrophoretically slow ferritin, which gave a major band corresponding to the L-subunit (20.7 kDa). Thus the electrophoretically fast ferritin resembles that reported by Massover [(1985) Biochim. Biophys. Acta 829, 377-386] in livers of mice with short-term parenteral iron overload. The electrophoretically fast ferritin had a lower iron content (2000 Fe atoms/molecule) than the electrophoretically slow ferritin (3000 Fe atoms/molecule). Removal and re-incorporation of iron was possible without effect on the electrophoretic mobility of either ferritin species. On subcellular fractionation the electrophoretically fast ferritin was enriched in pellet fractions and was the sole soluble ferritin isolated from iron-laden secondary lysosomes (siderosomes). The amount and relative proportion of the electrophoretically fast species increased with iron loading. Haemosiderin isolated from siderosomes was found to contain a peptide reactive to anti-ferritin serum and corresponding to the 17.3 kDa peptide of the electrophoretically fast ferritin species. Unlike the electrophoretically slow ferritin, the electrophoretically fast ferritin did not become significantly radioactive in a 1 h biosynthetic labelling experiment. We conclude that the minor ferritin is not, as has been suggested for mouse liver ferritin, 'a completely new species of smaller holoferritin that represents a shift in the ferritin phenotype' in response to siderosis, but a precursor of haemosiderin, in agreement with the proposal by Richter [(1984) Lab. Invest. 50, 26-35] concerning siderosomal ferritin.

Animals↗
PubMed1987

A new form of ferritin heterogeneity explained. Isolation and identification of a nineteen-amino-acid-residue fragment from siderosomal ferritin of rat liver.

Ferritin present within siderosomes of iron-loaded rats has a faster anodal mobility than that of cytosolic ferritin from the same rats. A 19-amino-acid-residue peptide was isolated from this fast ferritin and shown to be derived from the C-terminal end of its L-subunit. A 17.3 kDa peptide seen on electrophoresis in denaturing gels of this ferritin accounts for the major portion of the original 182-residue subunit. The two peptides arise from cleavage within the 'insertion region' of the L-subunit sequence that occurs between the D and E helices and lies on the outside of the assembled molecule. This cleavage is present in about 80% of the L-subunits of siderosomal ferritin but nevertheless leaves the molecular structure otherwise intact. It gives rise to an apparent decrease in molecular size, accounting for the faster anodal mobility on native gels. Hence a new form of heterogeneity in ferritin preparations has been explained.

Amino Acid Sequence↗
PubMed1986

Ferritin as an iron-storage protein: mechanisms of iron uptake.

The major physiological role of the ubiquitous protein ferritin is to store iron, which it does as a mineral core within a protein shell. Using evidence from a variety of sources we suggest that this versatile protein responds in its mechanism of iron uptake to changes in environmental conditions.

Ferritins↗
PubMed1984

The location of antigenic sites on ferritin molecules.

Immunoreactivities of peptides purified after cleavage of human liver apoferritin are reported and discussed in relation to the known 3-dimensional and primary structures of homologous apoferritins. These studies point to 3 antigenic sites occupying continuous inter-helical regions of the polypeptide chains which lie on the surface of the apoferritin molecule. Other antigenic regions may encompass amino acids remote in the primary structure or belonging to different subunits.

Animals↗
PubMed1984

Iron-induced changes in rat liver isoferritins.

The effects of single and of multiple iron injection on the distribution of isoferritins was studied in rat liver with the aid of 14C-labelling either after or before iron treatment. Several effects of iron can be seen. Analysis of protein and labelling patterns show that it not only produces a disproportionate increase in the more-basic isoferritins, but may, in sufficient dose, actually lead to a decrease in the more-acidic isoferritins. Use of iron injection after radioactivity shows that it must give rise to post-assembly changes causing acidic isoferritins to become more basic. With a moderate iron dose this change is relatively slow, taking several hours, and seems to occur in addition to a differential stimulation of the synthesis of the more-basic isoferritins. With higher iron dosage the post-assembly changes may be so rapid that it is difficult to distinguish them from a switch in the pattern of synthesis.

Animals↗
PubMed1984

Non-random distribution of iron entering rat liver ferritin in vivo.

Radioactive iron incorporated into rat liver ferritin in vivo and then released in vitro up to 24h after injection follows 'last-in-first-out' behaviour. Thus, within this period at least, the added iron does not equilibrate with iron already present in the molecule's iron-core.

Animals↗
PubMed1984

Functional studies on rat-liver isoferritins.

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.

Animals↗