Interaction of aluminium citrate with horse spleen ferritin.
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ABSTRACT:
SUBMITTER: Dedman DJ
PROVIDER: S-EPMC1133195 | biostudies-other | 1992 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Native electron capture dissociation (NECD) is a process during which proteins undergo fragmentation similar to that from radical dissociation methods, but without the addition of exogenous electrons. However, after three initial reports of NECD from the cytochrome c dimer complex, no further evidence of the effect has been published. Here, we report NECD behavior from horse spleen ferritin, a ∼490 kDa protein complex ∼20-fold larger than the previously studied cytochrome c dimer. Application of front-end infrared excitation (FIRE) in conjunction with low- and high-m/z quadrupole isolation and collisionally activated dissociation (CAD) provides new insights into the NECD mechanism. Additionally, activation of the intact complex in either the electrospray droplet or the gas phase produced c-type fragment ions. Similar to the previously reported results on cytochrome c, these fragment ions form near residues known to interact with iron atoms in solution. By mapping the location of backbone cleavages associated with c-type ions onto the crystal structure, we are able to characterize two distinct iron binding channels that facilitate iron ion transport into the core of the complex. The resulting pathways are in good agreement with previously reported results for iron binding sites in mammalian ferritin.
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Project description:When ferritin is reconstituted from Fe and apoferritin in vitro in the presence of Pi, the product obtained differs both from native ferritin and from ferritin reconstituted in the absence of Pi. When the latter is incubated with Pi the product resembles native ferritin with respect both to the pattern of Pi incorporated per molecule or per Fe atom and to the ease of release of this Pi relative to Fe release. It is concluded that much of the Pi of native ferritin is adsorbed on surfaces of ferritin iron-core crystallites. The results also suggest that Pi is not present at the intracellular site of Fe incorporation into ferritin, but is added after Fe.
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| S-EPMC5676598 | biostudies-literature
Project description:The rate of release of iron to 1,10-phenanthroline from ferritin fractions of different iron contents has been studied. The experimental results could be interpreted by a simple hypothetical model of the shape of the hydrous ferric oxide micelle at different iron contents, and reasonable correlation obtained between the rate of release and the calculated micelle surface areas. Initial rates of release did not correlate significantly with protein concentration.
| S-EPMC1166081 | biostudies-other
Project description:Ferritin-Fe(III) was rapidly and quantitatively reduced and liberated as Fe(II) by FMNH(2), FADH(2) and reduced riboflavin. Dithionite also released Fe(II) from ferritin but at less than 1% of the rate with FMNH(2). Cysteine, glutathione and ascorbate gave a similar slower rate and yielded less than 20% of the total iron from ferritin within a few hours. The reduction of ferritin-Fe(III) by the three riboflavin compounds gave complex second-order kinetics with overlapping fast and slow reactions. The fast reaction appeared to be non-specific and may be due to a reduction of Fe(III) of a lower degree of polymerization, equilibrated with ferritin iron. The amount of this Fe(3+) ion initially reduced was small, less than 0.3% of the total iron. Addition of FMN to the ferritin-dithionite system enhanced the reduction; this is due to the reduction of FMN by dithionite to form FMNH(2) which then reduces ferritin-Fe(III). A comparison of the thermodynamic parameters of FMNH(2)-ferritin and dithionite-ferritin complex formation showed that FMNH(2) required a lower activation energy and a negative entropy change, whereas dithionite required 50% more activation energy and showed a positive entropy change in ferritin reduction. The effectiveness of FMNH(2) in ferritin-Fe(III) reduction may be due to a specific binding of the riboflavin moiety to the protein portion of the ferritin molecule.
| S-EPMC1168386 | biostudies-other
Project description:Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results were consistent with the difference in calculated isoelectric points of chicken and horse ferritin subunits. By two-dimensional gel electrophoresis, chicken ferritin 22 kDa subunits exhibited isoelectric points from 6.1 to 6.6 whereas horse spleen ferritin subunits exhibited isoelectric points of 5.8-6.3. The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6 whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9. Intact chicken liver ferritin particles were 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in diameter when viewed by electron microscopy. Horse spleen ferritin consisted of slightly smaller particles with an average diameter of 11.0 +/- 0.7 nm. However, ferritin from chicken liver and horse spleen co-migrated with an apparent molecular mass of 470 kDa when analysed by Sepharose 4B gel filtration chromatography. These results indicate that, consistent with results from other published purification methods, the chicken ferritin purified by the methods reported here exhibits both structural similarities to, and differences from, horse spleen ferritin.
| S-EPMC1138378 | biostudies-other
Project description:1. Ferritin was isolated from human and horse spleen and liver, and apoferritin prepared therefrom. 2. The electrophoretic mobilities of the four apoferritins were determined on polyacrylamide gels and on cellulose acetate strips, and all found to be equal. 3. Homologous ferritins share reactions of identity in immunodiffusion experiments, whereas heterologous ferritins show only partial identity. 4. The subunit molecular weight of each of the apoferritins was determined by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate and by chromatography on agarose columns in 6m-guanidine-HCl. A value of approx. 18500 was found in all cases. The proteins all had sedimentation coefficients of 17-18S. It thus seems that they have identical quaternary structures. 5. The amino acid compositions of the proteins revealed distinct differences both between organs and between species. This was confirmed by analysis of the tryptic peptide patterns, where it was found that about one-third of the peptides were common to the four proteins and the other two-thirds varied from protein to protein. 6. It is concluded that the apoferritins present in the liver and spleen of human and horse are both organ- and species-specific. 7. The apoferritin isolated from the liver of a patient with idiopathic haemochromatosis was identical with normal human liver apoferritin by the criteria described above.
| S-EPMC1177438 | biostudies-other
Project description:Horse spleen ferritin is shown to bind haem to generate a haemoprotein, named herein haemoferritin. A total of 14-16 haem molecules are bound per 24 subunits of ferritin. The molecular mass of the non-haem-iron-free haemoferritin has been determined to be 420 +/- 40 kDa, indicating that haem binding does not lead to dissociation of the 24 subunits that comprise the ferritin molecule. The functional role of the bound haem has been investigated with respect to the release of iron from the non-haem iron core. The bound haem is shown to increase the rate of iron release in a reductive assay system. In the absence of haem the rate of iron release depends on the redox potential of the reductant, but in the presence of haem the rate is largely independent of the reductant and is faster than the rate for the haem-free ferritin. These data haem, but in the presence of haem electron transfer is not rate-limiting.
| S-EPMC1130867 | biostudies-other
Project description:BackgroundEucalyptus is the main plantation wood species, mostly grown in aluminized acid soils. To understand the response of Eucalyptus clones to aluminum (Al) toxicity, the Al-tolerant Eucalyptus grandis × E. urophylla clone GL-9 (designated "G9") and the Al-sensitive E. urophylla clone GL-4 (designated "W4") were employed to investigate the production and secretion of citrate and malate by roots.ResultsEucalyptus seedlings in hydroponics were exposed to the presence or absence of 4.4 mM Al at pH 4.0 for 24 h. The protein synthesis inhibitor cycloheximide (CHM) and anion channel blocker phenylglyoxal (PG) were applied to explore possible pathways involved in organic acid secretion. The secretion of malate and citrate was earlier and greater in G9 than in W4, corresponding to less Al accumulation in G9. The concentration of Al in G9 roots peaked after 1 h and decreased afterwards, corresponding with a rapid induction of malate secretion. A time-lag of about 6 h in citrate efflux in G9 was followed by robust secretion to support continuous Al-detoxification. Malate secretion alone may alleviate Al toxicity because the peaks of Al accumulation and malate secretion were simultaneous in W4, which did not secrete appreciable citrate. Enhanced activities of citrate synthase (CS) and phosphoenolpyruvate carboxylase (PEPC), and reduced activities of isocitrate dehydrogenase (IDH), aconitase (ACO) and malic enzyme (ME) were closely associated with the greater secretion of citrate in G9. PG effectively inhibited citrate and malate secretion in both Eucalyptus clones. CHM also inhibited malate and citrate secretion in G9, and citrate secretion in W4, but notably did not affect malate secretion in W4.ConclusionsG9 immediately secrete malate from roots, which had an initial effect on Al-detoxification, followed by time-delayed citrate secretion. Pre-existing anion channel protein first contributed to malate secretion, while synthesis of carrier protein appeared to be needed for citrate excretion. The changes of organic acid concentrations in response to Al can be achieved by enhanced CS and PEPC activities, but was supported by changes in the activities of other enzymes involved in organic acid metabolism. The above information may help to further explore genes related to Al-tolerance in Eucalyptus.
| S-EPMC7789223 | biostudies-literature
Project description:Reactions of reduced horse spleen ferritin with horse and Saccharomyces cerevisiae ferricytochromes c, cow ferricytochrome b5, sperm-whale metmyoglobin and Pseudomonas aeruginosa ferricytochrome c-551 were investigated by u.v.-visible spectrophotometry. In all cases the reduced ferritin reduced the ferrihaemoproteins. The rate of reduction varied from less than 0.2 M-1.s-1 for metmyoglobin to 1.1 x 10(3) M-1.s-1 for horse ferricytochrome c (0.1 M-phosphate buffer, pH 7.4, at 25 degrees C). We conclude that the mechanism of ferrihaemoprotein reduction involves long-range electron transfer through the coat of ferritin and that such electron transfer is rapid enough to account for the rates of iron release observed by other workers in reductive release assays.
| S-EPMC1151419 | biostudies-other