High-resolution preparative electrochromatography for purification of two subunit types of ferritin

Otsuka, Soichi; Listowsky, Irving

doi:10.1016/0003-2697(80)90176-1

Cited by 21 publications

(12 citation statements)

References 12 publications

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“…The two subunits have similar amino acid compositions and peptide sequences in common, and both are synthesized in cell-free systems by using unfractionated rat liver mRNA' (Arosio et al, 1978). We have recently described methods for fractionation and purification of these two structurally similar subunits (Otsuka & Listowsky, 1980). In this report homogeneous H and L subunits of human liver ferritin are characterized.…”

Section: Resultsmentioning

confidence: 99%

“…The protein was dissociated into subunits by incubation in 9 M urea, pH 2.5, for 10 min, readjustment of the pH to 7.0, and centrifugation at 57000g for 2 h to pellet the inorganic iron micelles (Listowsky et al, 1972). Homogeneous H-and L-type subunits were prepared by the electrochromatography procedures described previously (Otsuka & Listowsky, 1980). For reassembly of discrete (H-and L-containing) apoferritin components, subunit solutions (0.5-1 .O mg/mL) in 9 M urea were dialyzed successively into 4 M urea, 2 M urea, and 0.01 M sodium phosphate buffer, pH 7.0, in the presence of 1 mM dithiothreitol.…”

Section: Methodsmentioning

confidence: 99%

“…The protein was therefore incubated in 9 M urea at pH 2.5 for 10 min and the pH readjusted to 7.0 these conditions promote its dissociation into subunits (Listowsky et al, 1972). Homogeneous H and L subunit types were prepared by electrochromatography procedures (Otsuka & Listowsky, 1980). After removal of urea by stepwise dialysis procedures (cf.…”

Section: Electron Microscopymentioning

confidence: 99%

“…The antisera may also be used to support the notion that natural ferritin is a true hybrid system with individual molecules containing both subunits (Otsuka et al, 1980). It had been suggested that tissue ferritins are mixtures of two homopolymers and give rise to families of proteins by minor posttranslational modifications .…”

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confidence: 99%

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Structure, assembly, conformation, and immunological properties of the two subunit classes of ferritin

Otsuka¹,

Maruyama²,

Listowsky³

1981

Biochemistry

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The two subunit types of human liver ferritin were purified to homogeneity. Both subunits reassembled in a well-defined manner and formed spherical particles that resembled natural apoferritin in electron micrographs. Affinity chromatography methods were employed to obtain preparations of antibodies that interacted exclusively either with the H or with the L polypeptides, demonstrating that distinct immunological properties may be ascribed to each subunit of ferritin. The amino acid compositions of the subunits were similar, but the larger H subunit had fewer leucine, phenylalanine, and arginine residues. It is therefore improbable that H subunits undergo proteolytic processing and are precursors for L subunits. Circular dichroism data indicated that homopolymers assembled from L-type subunits had substantially more ordered secondary structures and greater alpha-helical contents than their H counterparts. Small differences in the environment of tryptophan residues were evident from fluorescence spectra of each homopolymer. In isoelectric focusing experiments reassembled H or L homopolymers migrated as families of proteins within discrete pI ranges which are probably representative of subpopulations of each subunit type. The H homopolymer focused at lower pI's than the L component. These data substantiate the contention that both subunits are authentic polypeptide moieties of ferritin with some common structural features, but the results also underscore prominent dissimilarities in their properties.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Electron Microscopymentioning

confidence: 99%

mentioning

confidence: 99%

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Structure, assembly, conformation, and immunological properties of the two subunit classes of ferritin

Otsuka¹,

Maruyama²,

Listowsky³

1981

Biochemistry

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“…Electrochromatography is a developing technique [l-81 which currently can be divided into two categories: electrochromatography with [ 1,2,4,5] and without [3,7,8] pressurized flow. In the latter case, electroosmosis is the driving force which carries the solute from column inlet to outlet.…”

Section: Introductionmentioning

confidence: 99%

Effects of pH and organic solvent on chromatographic behavior in capillary electrochromatography

Kitagawa

Tsuda

1994

J Microcolumn Separations

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Abstract. Electrochromatography is demonstrated using a 50 pm i.d. capillary column. The separation of solutes due to their different mobilities is obtained by the application of high voltage along the column. The elution times of solutes are dependent on the pH and composition of the eluent. Both electrophoretic and electroosmotic flow velocities are almost constant between 30 and 90% methanol in the eluent, and they increase with more than 90% methanol. The pH dependence of the electroosmotic flow velocity may be related to the dissociation of unreacted silanol groups on the surface of the ODs-silica particles.

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Introduction and Summary

Tsuda¹

1995

Electric Field Applications

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High-resolution preparative electrochromatography for purification of two subunit types of ferritin

Cited by 21 publications

References 12 publications

Structure, assembly, conformation, and immunological properties of the two subunit classes of ferritin

Structure, assembly, conformation, and immunological properties of the two subunit classes of ferritin

Effects of pH and organic solvent on chromatographic behavior in capillary electrochromatography

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