Reversible dissociation of ferritin and its subunits in vitro

Smith-Johannsen, Heidi; Drysdale, James W.

doi:10.1016/0005-2795(69)90177-9

Cited by 32 publications

(19 citation statements)

References 12 publications

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“…This was performed using an Ortec (Ortec Iiicorporation, Oak Ridge, Tennessee) electrophoresis system which provides for multiple parallel samples 011 onc gel slab thus allowing precise comparison betwceii samples. Electrophoresis was performed in a 4.5% polyacrylamide gel with 4% iiicthylaiebisacrylamide cross-linker in 0.065 M trisborate buffer pH 9.0 at 4°C and a constant voltage of 100 V. Thc gcl slab was stained for protein using Poiiccau S (Smith-Johaniisen & Drysdale, 19691, or Coomassie blue and counter-stained for iron with potassium ferrocyanide (Smith-Johaiinsen & Drysdale, 1969).…”

Section: Methodsmentioning

confidence: 99%

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Human Isoferritins: Organ Specific Iron and Apoferritin Distribution

et al. 1975

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Ferritins from human liver, spleen, heart, pancreas and kidney were compared by electrophoresis and isoelectric focusing in polyacrylamide gels, by immunodiffusion against antisera to homologous and heterologous ferritin, and in some cases by their cyanogen bromide peptides. All ferritins appeared to consist of a single species on gel electrophoresis with the exception of heart ferritin which separated into two major components. Small differences in electrophoretic mobility were found in all tissue ferritins. By contrast, all tissue ferritins were found to consist of multiple forms when analysed by gel electrofocusing. At least five isoferritins were found in most tissues, several of which were common to most tissues. At least two were common to all tissues. Those ferritins which were most easily distinguishable electrophoretically, e.g. spleen and heart ferritin, showed the greatest differences on gel electrofocusing. The ferritin profile was characteristic of each organ and was reproducible both within individuals and between individual tissues. There were striking differences in the iron content of the various isoferritins within a tissue. Further, the iron content of isoferritins common to more than one tissue varied with the tissue of origin. Some isoferritins in several organs and all of the isoferritins in pancreas appeared to contain little, if any, iron. All five tissue ferritins contained antigenic determinants in common with liver ferritin. However, an additional antigenic determinant was found in liver ferritin which was not detectable in the ferritins from the other organs.

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Section: Methodsmentioning

confidence: 99%

“…of the iron core (Drysdale et a f , 1968;Harrison & Gregory, 1968;Smith-Johannsen & Drysdale, 1969). Apofcrritin is presently thought to consist of 20 or 24 chemically identical subunits (Crichton, 1971), but recent evidence of multiple subunit types (Linder & Munro, 1973 ;Niitsu et al, 1973) indicates a considerably more complex structure.…”

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Human Isoferritins: Organ Specific Iron and Apoferritin Distribution

et al. 1975

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“…The H and L subunits are isomorphous and share the same tertiary structure with a bundle of four antiparallel α-helices, a shorter helix on top of them (see Fig. 1), and loops connecting the helices (Stefanini et al, 1996;Massover, 1993). The subunits are roughly cylindrical, a little more than 5 nm long and 2.5 nm wide.…”

Section: Ferritin and Apoferritinmentioning

confidence: 99%

“…Apoferritin is obtained from ferritin by removing the iron oxide. Horse spleen apoferritin consists of 85 %-90 % L and 10 %-15 % H chains (Stefanini et al, 1996;May et al, 2010). From sedimentation velocity measurements molar masses of 440-500 kDa (Thomas et al, 1998;May et al, 2010;Ghirlando et al, 2016) are estimated for apoferritin while the calculation based on the subunit molar masses determined from cDNA sequences (H type: 21 269 Da, L type: 19 978 Da) yields 481.2 kDa (assuming 85 % L and 15 % H chains).…”

Section: Ferritin and Apoferritinmentioning

confidence: 99%

Protein aggregates nucleate ice: the example of apoferritin

et al. 2020

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Abstract. Biological material has gained increasing attention recently as a source of ice-nucleating particles that may account for cloud glaciation at moderate supercooling. While the ice-nucleation (IN) ability of some bacteria can be related to membrane-bound proteins with epitaxial fit to ice, little is known about the IN-active entities present in biological material in general. To elucidate the potential of proteins and viruses to contribute to the IN activity of biological material, we performed bulk freezing experiments with the newly developed drop freezing assay DRoplet Ice Nuclei Counter Zurich (DRINCZ), which allows the simultaneous cooling of 96 sample aliquots in a chilled ethanol bath. We performed a screening of common proteins, namely the iron storage protein ferritin and its iron-free counterpart apoferritin, the milk protein casein, the egg protein ovalbumin, two hydrophobins, and a yeast ice-binding protein, all of which revealed IN activity with active site densities > 0.1 mg−1 at −10 ∘C. The tobacco mosaic virus, a plant virus based on helically assembled proteins, also proved to be IN active with active site densities increasing from 100 mg−1 at −14 ∘C to 10 000 mg−1 at −20 ∘C. Among the screened proteins, the IN activity of horse spleen ferritin and apoferritin, which form cages of 24 co-assembled protein subunits, proved to be outstanding with active site densities > 10 mg−1 at −5 ∘C. Investigation of the pH dependence and heat resistance of the apoferritin sample confirmed the proteinaceous nature of its IN-active entities but excluded the correctly folded cage monomer as the IN-active species. A dilution series of apoferritin in water revealed two distinct freezing ranges, an upper one from −4 to −11 ∘C and a lower one from −11 to −21 ∘C. Dynamic light scattering measurements related the upper freezing range to ice-nucleating sites residing on aggregates and the lower freezing range to sites located on misfolded cage monomers or oligomers. The sites proved to persist during several freeze–thaw cycles performed with the same sample aliquots. Based on these results, IN activity seems to be a common feature of diverse proteins, irrespective of their function, but arising only rarely, most probably through defective folding or aggregation to structures that are IN active.

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“…then chilled and added in equal amount to native ferritin. which, in these conditions, does not dissociate (Johannsen & Drysdale. 1969): the mixtures were run on PAGE made by 15% acrylamide on the bottom part, and 7.5% acrylamide on the top part.…”

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confidence: 99%

Antibodies for denatured human H‐ferritin stain only reticuloendothelial cells within the bone marrow

et al. 1992

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Human H-ferritin homopolymer was denatured in sodium dodecyl sulphate and injected in mice to obtain antibodies for dissociated H-subunit. The antisera and Moabs obtained were specific for the denatured H-chain with no cross-reactivity with assembled ferritins in immunoblotting experiments. In contrast the Moabs for native recombinant H-ferritin are specific for the assembled ferritin molecules with weak cross-reactivity with the denatured H-subunits. The epitope recognized by one of the anti-denatured H-chain Moabs was mapped on the C-terminal helix of ferritin. The antibodies were used to study H-ferritin conformation in cells. In immunocytochemistry experiments the antibodies for denatured H-ferritin stained HeLa and K562 cells weakly, with a different intensity and pattern to those obtained with anti-native H-ferritin antibody. In human bone marrow smears the anti-denatured ferritin antibodies stained only reticuloendothelial cells, and did not recognize the H-ferritin rich immature erythroblasts. It is concluded that assembled and denatured H-ferritins are immunogenically distinct, and that erythroid and reticuloendothelial cells within the bone marrow contain H-ferritin in different conformations.

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Reversible dissociation of ferritin and its subunits in vitro

Cited by 32 publications

References 12 publications

Human Isoferritins: Organ Specific Iron and Apoferritin Distribution

Human Isoferritins: Organ Specific Iron and Apoferritin Distribution

Protein aggregates nucleate ice: the example of apoferritin

Antibodies for denatured human H‐ferritin stain only reticuloendothelial cells within the bone marrow

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