Comparison of the physical properties of chemically prepared and tissue-endogenous equine apoferritins.

Leach, Bonnie Strayer; May, Michael; Fish, Wayne W.

doi:10.1016/s0021-9258(17)33326-4

Cited by 21 publications

(4 citation statements)

References 22 publications

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“…Gel filtration, electrophoretic, spectroscopic, and crystallization data were analogous for rLFo and HLF, suggesting a structural similarity which was confirmed by the CD analyses. The CD spectra of the two proteins in the farand near-UV regions were nearly identical, and in good agreement with previous studies on human and horse ferritins (Leach et al, 1976; Listowsky et al, 1972). The high molar ellipticity values at 222 nm are an index of correct folding of the sub-units, while the positive ellipticity values in the near-UV region have been interpreted as an index of correct ferritin assembly (Leach et al, 1976;Stefanini et al, 1982).…”

Section: Discussionsupporting

confidence: 88%

Expression and structural and functional properties of human ferritin L-chain from Escherichia coli

Levi¹,

et al. 1989

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The human ferritin L-chain cDNA was cloned into a vector for overproduction in Escherichia coli, under the regulation of a lambda promoter. The plasmid obtained contains the full L-chain coding region modified at the first two codons. It is able to direct the synthesis of the L-chain which can constitute up to 15% of the total soluble protein of bacterial extract. The L-chains assemble to form a ferritin homopolymer with electrophoretic mobility, molecular weight, thermal stability, spectroscopic, and immunological properties analogous to natural ferritin from human liver (95% L-chain). This recombinant L-ferritin is able to incorporate and retain iron in solution at physiological pH values. At variance with the H-ferritin, the L form does not uptake iron at acidic pH values and does not show detectable ferroxidase activity. It is concluded that ferritin L-chain lacks the ferroxidase site present in the H-chain and that the two chains may have specialized functions in intracellular iron metabolism.

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Section: Discussionsupporting

confidence: 88%

Expression and structural and functional properties of human ferritin L-chain from Escherichia coli

Levi¹,

et al. 1989

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“…The gels were stained for iron with Prussian Blue. 1976; Leach et al, 1976). In contrast, heart apoferritin shows two positive bands at 293 and 286 nm and a negative absorption between 283 and 255 nm with peaks at 268 and 262 nm.…”

Section: Resultsmentioning

confidence: 98%

“…The dissociated subunits of all apoferritins exhibit a relatively weak negative CD spectrum in the tyrosyl and phenylalanyl region, but no tryptophanyl peaks. All these and the previously available spectroscopic data (Stefanini et al, 1976b;Leach et al, 1976; Crichton & can be interpreted by assuming that both subunit types can exist in two conformations. The transition between the "unlocked" conformation characteristic of the monomer and the "locked" conformation characteristic of the polymer occurs in the assembly process.…”

Section: Discussionmentioning

confidence: 99%

Structural heterogeneity and subunit composition of horse ferritins

Stefanini¹,

Chiancone²,

Arosio³

et al. 1982

Biochemistry

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Structural, spectroscopic, and immunological properties of horse ferritins extracted from spleen, liver, and heart were studied to test the hypothesis that the different tissue ferritins are hybrids composed of variable proportions of two subunit types. The weight-average molecular weights determined by sedimentation velocity and gel filtration increase from 460 000 for spleen to 480 000 for liver and to 515 000 for heart apoferritin; moreover, the diffusion coefficients prove that each tissue-specific ferritin consists of a population of hybrid molecules. The intrinsic fluorescence and the near-UV circular dichroism (CD) spectra change as a function of the subunit composition of the three ferritins. The fluorescence emission maximum, which occurs at a very low wavelength (315 nm) in horse spleen apoferritin, is shifted to increasingly higher wavelengths in the liver and heart proteins (320 and 325 nm, respectively), indicating that the tryptophan and tyrosine residues become less rigidly immobilized with an increase in the H-subunit content. The tryptophan residues behave as fully solvated in the monomeric subunits at acidic pH values. In accordance with the fluorescence data, the near-UV CD spectra show that the tryptophan environment is highly asymmetric in spleen apoferritin, progressively less so in liver and heart apoferritins, and completely relaxed in the dissociated subunits. Moreover, they show that the environment of tyrosines and phenylalanines differs markedly in the spleen and heart apoproteins. The ferritins studied appear to have immunogenic sites which are specific for the H and L subunits on the basis of enzyme-linked immunoassay and double-diffusion experiments.

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“…(Stefanini et al, 1982); apoferritin was obtained by reduction of iron with sodium dithionite and chelation with , '-bipyridyl (Stefanini et al, 1975). The quality of the apoferritin preparations was checked by means of their CD spectra in the near-UV region (Leach et al, 1976); their iron content was determined as the , '-bipyridyl complex at 520 nm (Bothwell et al, 1955) and was found to correspond to about five iron atoms per molecule. The apoferritin concentration was calculated from the absorption at 280 nm by using the extinction coefficient £¡*m = 9.0 (Bryce & Crichton, 1973) or by the Bradford method (Bradford, 1976).…”

Section: Methodsmentioning

confidence: 99%

Identification of the iron entry channels in apoferritin. Chemical modification and spectroscopic studies

Stefanini¹,

Desideri²,

Vecchini³

et al. 1989

Biochemistry

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The knowledge of the route through which iron can enter and leave the apoferritin shell is a prerequisite for the understanding of ferritin's function. The involvement of the hydrophilic 3-fold channels in the iron uptake process has been studied by taking advantage of the reactivity of specific residues that line such channels, i.e., glutamic acid-127 and aspartic acid-130, the major Cd(II) binding sites, and cysteine-126. 113Cd NMR experiments have provided direct evidence for the competition between Fe(II) and Cd(II) binding to major Cd(II) binding sites on the protein and or a higher affinity of Fe(II) for these sites, in line with the well-known inhibitory effect of Cd(II) on iron uptake. Further evidence for the use of the 3-fold channels in the iron entry process has been obtained by means of chemical modification of Cys-126 with different mercurials. In particular, the introduction of the additional carboxylate carried by p-(chloromercuri)benzoate near Asp-127 and Glu-130 increases the initial rate of iron uptake and affects the coordination geometry of the metal in the Fe(III)-apoferritin complex as indicated by optical absorption and EPR data. The assignment of these effects to the carboxylate moiety of p-(chloromercuri)benzoate is brought out by the observation that the introduction in the 3-fold channel of the benzene ring only by means of phenylmercuric acetate has no effect on the initial iron uptake kinetics and on the spectroscopic properties of the Fe(III)-apoferritin complex.

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Comparison of the physical properties of chemically prepared and tissue-endogenous equine apoferritins.

Cited by 21 publications

References 22 publications

Expression and structural and functional properties of human ferritin L-chain from Escherichia coli

Expression and structural and functional properties of human ferritin L-chain from Escherichia coli

Structural heterogeneity and subunit composition of horse ferritins

Identification of the iron entry channels in apoferritin. Chemical modification and spectroscopic studies

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