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Query: UNIPROT:P02794 (ferritin)
 17,525 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Soluble and insoluble forms of ferritins have been purified from dry pea seeds by gel filtration. The insoluble form is called phytosiderin by analogy with animal hemosiderin. Native gel electrophoresis of these two forms have shown that the soluble one (ferritin) is homogenous in size and more compact than the insoluble one (phytosiderin) which is heterogenous in size. However, when iron is removed from these two classes of molecules (apoferritin), they have the same mobility in isopycnic centrifugations. Polyacrylamide-sodium dodecyl sulfate gel electrophoresis revealed a difference in their subunit composition: ferritin molecules are built up from a 28-kDa subunit and phytosiderin from a 26.5-kDa subunit. Partial proteolysis using a Staphylococcus aureus protease indicates a strong relationship between these two polypeptides. Intermediates between these two forms have also been characterized and are composed of both subunits in various amounts. Ferritin and phytosiderin are both able to incorporate iron in vitro into their mineral core. It is also shown that in vitro iron exchange induces ferritin degradation. This degradation is prevented by inhibitors of the Fenton cycle (iron chelates like o-phenanthroline and desferrioxamine B) and reduced by Tris, a radical scavenger. Under in vitro conditions of controlled radical damage the 28-kDa subunit is converted into the 26.5-kDa subunit. Purification of the 28-kDa subunit has allowed us to determine the NH2-terminal sequence. The NH2 extremity of the 26.5-kDa subunit is heterogenous, but the sequence of its main component is identical to the sequence of the 28-kDa subunit downstream residue Leu-21. These data indicate that the 26.5-kDa subunit is produced by radical mediated damage leading to a series of cleavages in the NH2 terminal part of the 28-kDa subunit.
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PMID:Mechanism of the transition from plant ferritin to phytosiderin. 253 54

The iron-storage protein ferritin was found to be associated with highly purified coated vesicles (CV) from chicken liver. Chicken liver ferritin was morphologically similar to ferritin from horse spleen and could be isolated using a specific anti-ferritin monoclonal antibody. This antibody recognized a 240 X 10(3) Mr form of chicken ferritin but not the 22 X 10(3) Mr ferritin subunit after protein transfer to nitrocellulose. CV purified by controlled-pore glass-bead chromatography also contained ferritin when assayed by monoclonal anti-ferritin antibody using a sensitive enzyme-linked assay. Ferritin remained associated with CV even after re-chromatography. Ferritin particles were observed to be associated with CV by electron microscopy. CV-associated ferritin could be quantitatively removed from CV by treatment of the CV with 0.5 M-Tris-HC1 + 2M-urea at pH 8.5, conditions that also lead to dissociation of the clathrin lattice. Triton X-100 detergent treatment did not affect the association of ferritin with CV. These results indicate that purified CV from chicken liver contain ferritin in association with the clathrin lattice. The possible functional significance of this association is discussed.
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PMID:Coated vesicles from chicken liver bind ferritin. 277 20

An optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5% T, 3% C, were internally stabilized with the aid of an AcrylAide-pretreated, hydrophilized polyester fabric, preferably with a 60 microns mesh opening. Ultrathin (120-180 microns) gels were prepared with the flap technique and 500 microns gels with the cassette technique; 500 microns gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1-3% w/v carrier ampholytes. Incorporation of 40-60% w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900-1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1-0.2 micron pore size nitrocellulose membranes in 10-20 min from ultrathin gels and in 30-60 min from 500 microns gels. Based on quantification of residual protein in the gels after blotting, a transfer efficiency of 60-87% was found for the ultrathin and 53-69% for the 500 microns gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris-glycine buffer system electrophoretic blotting required only 2-5 min with ultrathin gels and 20 min with 500 microns gels. Marker proteins, including horse spleen ferritin (Mr465,000), could be transferred with 91-96% efficiency.
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PMID:Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients. 324 47

The translational diffusion coefficient D020,w, of monomeric human immunoglobulin G (IgG) has been studied by photon-correlation spectroscopy as a function of pH and protein concentration. At pH 7.6, we find D020,w = 3.89 x 10(-7) cm2/sec, in good agreement with the value determined by classic methods. This value corresponds to an effective hydrodynamic radius R, of 55.1 +/- 0.3 A. As pH is increased to 8.9; with the same ionic strength, the molecule appears to expand slightly (3.5% increase in hydrodynamic radius). The concentration dependence of the IgG diffusion constant is interpreted in terms of solution electrostatic effects and shows that long-range repulsive interactions are negligible in the buffer used. The diffusion coefficient for dimeric IgG has also been determined to be D20,w = 2.81 x 10(-7) +/- 0.04 cm2/sec at 1.6 mg/ml, which corresponds to a hydrodynamic radius of 75 A. For light-scattering studies of protein molecules in the dimension range of 5-10 nm (Mr approximately 10(5)-10(7] we find monomeric horse spleen ferritin well suited as a reference standard. Ferritin is a spherical molecule with a hydrodynamic radius R of 6.9 +/- 0.1 nm and is stable for years in our standard Tris-HCl-NaCl buffer even at room temperature.
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PMID:Photon correlation spectroscopy of human IgG. 325 67

Sporozoites of Eimeria tenella were incubated for 10, 20, or 30 min with parasite-specific monoclonal IgG antibody 3D3II from mice and then rinsed in a Tris-buffered glucose saline solution (TBGS). Some sporozoites were then incubated for 10, 20, or 30 min with ferritin- or colloidal gold-conjugated goat anti-mouse IgG antibody and then fixed in 2.5% glutaraldehyde and prepared for transmission (TEM) or scanning (SEM) electron microscopy. Other sporozoites that had been previously exposed to monoclonal antibody were prefixed with 0.25% glutaraldehyde, incubated with ferritin- or colloidal gold-conjugated anti-mouse IgG antibody and then fixed and prepared for TEM or SEM. Control preparations consisted of sporozoites exposed only to TBGS, monoclonal antibody 3D3II or to ferritin- or colloidal gold-conjugated anti-mouse IgG antibody. Capping of immune complexes occurred only on the surface of those sporozoites exposed to monoclonal antibody 3D3II followed by ferritin- or gold-conjugated antibody. Immune complexes moved laterally and posteriorly on the outer surface of the parasite plasma membrane to form a cap at the posterior end of the sporozoite. Capping did not occur in TBGS controls nor in sporozoites treated with monoclonal antibody 3D3II and prefixed in 0.25% glutaraldehyde before exposure to ferritin- or gold-conjugated antibody. Thus, capping of surface antigens did not occur in the presence of monoclonal 3D3II antibody only, whereas specimens exposed to both monoclonal and ferritin- or colloidal gold-conjugated antibodies were able to cap immune complexes.
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PMID:Capping of immune complexes by sporozoites of Eimeria tenella. 398 46

Canine marrow cells were incubated with transferrin-bound (59)Fe, and the partition of cellular iron was studied by chromatographic and gel filtration methods. Splitting-off of iron from the stromal fraction was avoided by lysing the cells in Tris HCl buffer at pH 8.6. Cellular iron was divided into four major compartments: stroma, microsomes, main hemoglobin, and fraction I. The iron in fraction I was found in ferritin, heme proteins, and low molecular weight iron. With incubation times of 3-10 min, (59)Fe appeared promptly in the main hemoglobin. The entry of (59)Fe into ferritin paralleled that of hemoglobin but was smaller in amount. When the marrow cells were incubated with (59)Fe for 15-20 min and reincubated without radioactive iron, movement of (59)Fe into main hemoglobin was observed, and essentially all this iron came from the particulate fraction (stroma, mitochondria, and microsomes). In these chase experiments there was no change in the total quantity of (59)Fe in ferritin. There was no evidence of a significant hemoglobin precursor other than low molecular weight iron. DEPENDING UPON CONCENTRATION, LEAD WAS OBSERVED TO INHIBIT CELLULAR IRON METABOLISM AT SEVERAL POINTS: uptake of iron by the cell, movement of iron from stroma to the soluble intracellular compartment, and synthesis of hemoglobin. The most pronounced inhibitory effect of lead was always on hemoglobin synthesis with an increase in ferritin: hemoglobin ratio. Bipyridine appeared to trap intracellular ferrous iron and to inhibit synthesis of both hemoglobin and ferritin. It was concluded that iron moves from the stroma into the soluble intracellular compartment as low molecular weight iron, probably as a complex of ferrous iron with low molecular weight components of the cytoplasm, that serves as the source of iron for both hemoglobin and ferritin synthesis.
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PMID:Studies on the partition of iron in bone marrow cells. 496 2

A new primary fixative, ethyldimethylaminopropyl carbodi-imide-glutaraldehyde-Tris, has been combined with the use of saponin for membrane permeabilization to yield a procedure which preserves ultrastructural morphology, yet retains a cytoplasmic matrix permeable to globulin molecules. This allows pre-embedding localization of intracellular protein antigens in cultured cells by fluorescence or electron microscopy. A further combination of these methods with the 'ferritin bridge' techniqe has allowed discrete localization which is quantifiable. Together, these methods yield an overall technique which provides high quality ultrastructural morphological preservation and precise antigen localization. Examples of the localization of alpha 2-macroglobulin, actin, SV40 T-antigen, tubulin and p60src are demonstrated. Extension of these methods from cultured cells to intact tissue should be possible without major changes.
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PMID:Electron microscopic immunocytochemical localization of intracellular antigens in cultured cells: the EGS and ferritin bridge procedures. 616 Jan 23

Rat liver homogenates in 0.1 M Tris, pH 7.5, were heated to 80 degrees C, cooled immediately, and centrifuged at 24,000 X g, and 7Be2+ was added to the supernatant. Twenty-five per cent of the radioactivity was bound to a single protein. It was purified to homogeneity and identified to be ferritin as judged by different criteria. These were sucrose density gradient centrifugation, electrophoresis in polyacrylamide gel of the native or sodium dodecyl sulfate-treated protein, reactivity to antibodies, isoelectric focusing, and total amino acid composition. Comparative study of the ability of ferritin or apoferritin to bind Cd2+, Zn2+, Cu2+, and Be2+ was conducted by using a gel equilibrium technique, Centifree micropartition technique, and microcentrifuge desalting technique. Ferritin could be saturated with Cd2+ or Zn2+ or Cu2+ but not with Be2+ even after 800 g atoms of Be2+ were bound. None of the bound Be2+ was dialyzable at 4 degrees C in 0.05 Tris acetate buffer, pH 8.5, but at pH 6.5 over 80% of the bound metal ion was dialyzed after 72 h. By contrast, apoferritin bound similar amounts of all four metal ions, some of which were dialyzable. By spectrophotometric titrations at pH 6.5 of Be2+ with sulfosalicylic acid (SSA), BeKDSSA was calculated to be 5.0 X 10(-6) M and by competition of sulfosalicyclic acid and ferritin for Be2+ the BeKDferritin was calculated to be 6.8 X 10(-6) M.
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PMID:Ferritin. Binding of beryllium and other divalent metal ions. 641 22

Our previous work showed that immunization of mice with Schistosoma japonicum (Sj) immature eggs induced significant immunity against fecundity and embryonation of the parasite. The Sj adult cDNA library was screened by sera from rabbits against Sj immature egg antigen (RASjIEA). The genes encoding molecules which may induce immunity against fecundity/embryonation were chosen for further cloning and expression. First of all, RASjIEA was absorbed with E. coli lysate to remove cross reactive antibodies. The cDNA library was then immunoscreened using the routine method. The resulted positive plaques were rescreened till individual clones were confirmed. Phagemids were obtained using in vivo excision. The positive clones were amplified using PCR. The sizes of the genes were determined by agarose gel electrophoresis. After DNA sequencing of the genes cloned, Gene bank was searched and six different genes were identified from a total of 102 positive clones. One of six identified genes, Sj ferritin (SjFer) was chosen to subclone into pGMC vector. According to DNA sequences of Sj Fer and MCS (multiple cloning site) of the vector, forward primer (Fer/GMC1) and reverse primer (Fer/GMC2) were designed and used to amplify Sj Fer by PCR. The Sj Fer cDNA and expression vector pGMC were digested with BamHI and XhoI. The digested cDNA and pGMC were ligased by T4 DNA ligase to construct a recombinant which was then used to transform E. coli strain ER2566. The fusion protein GMCSF-Sj Ferritin was expressed in insoluble form, the inclusion body. Pellets were harvested and resolved in Tris-HCl buffer containing 8M urea. GMCSF-Sj Ferritin was purified by affinity chromatography using Ni-NTA resin. The molecular weight was determined by SDS-PAGE. This study first reports the gene encoding S. japonicum ferritin as a new candidate for schistosome vaccine.
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PMID:[Schistosoma japonicum ferritin: cloning, nucleotide sequencing, expression, and purification]. 1068 50

Iron incorporation by bovine spleen apoferritin either with ferrous ammonium sulfate in different buffers or with ferrous ammonium sulfate and phosphate was studied. Iron uptake and iron autoxidation were recorded spectrophotomerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependent iron autoxidation, with Tris showing less iron autoxidation than Hepes. An Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes was a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and of Hepes after the addition of phosphate showed a straight line. Phosphate accelerated the iron uptake rate. The iron loading kinetics of apoferritin in Hepes was dependent on apoferritin concentration. The Km value obtained from iron uptake kinetics was 4.5 microM, corresponding to the physiological iron concentration. These results demonstrate that iron loading of apoferritin was accomplished at physiological iron concentrations, which is essential for iron uptake, via two uptake pathways of dependent on iron concentration.
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PMID:Two pathways of iron uptake in bovine spleen apoferritin dependent on iron concentration. 1186 23


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