scholarly journals Ferroxidase activity of ferritin: effects of pH, buffer and Fe(II) and Fe(III) concentrations on Fe(II) autoxidation and ferroxidation

1999 ◽  
Vol 338 (3) ◽  
pp. 615-618 ◽  
Author(s):  
Xiaoke YANG ◽  
N. Dennis CHASTEEN
Keyword(s):  
Ph Control ◽  
Alternative Mechanism ◽  
Experimental Conditions ◽  
Iron Storage ◽  
Ferroxidase Activity ◽  
Effects Of Ph ◽  
Ph Stat ◽  
Ph Buffer ◽  
Horse Spleen Ferritin

It is widely accepted that iron deposition in the iron storage protein ferritin in vitro involves Fe(II) oxidation, and that ferritin facilitates this oxidation at a ferroxidase site on the protein. However, these views have recently been questioned, with the protein ferroxidase activity instead being attributed to autoxidation from the buffer alone. Ligand exchange between another protein with ferroxidase activity and ferritin has been proposed as an alternative mechanism for iron incorporation into ferritin. In the present work, a pH stat apparatus is used to eliminate the influence of buffers on iron(II) oxidation. Here we show that the recent experiments questioning the ferroxidase activity of ferritin were flawed by inadequate pH control, that buffers actually retard rather than facilitate iron(II) oxidation, and that horse spleen ferritin has ferroxidase activity when measured under proper experimental conditions. Furthermore, high pH (7.0), a high Fe(II) concentration and the presence of Fe(III) all favour Fe(II) autoxidation in the presence or absence of ferritin.

scholarly journals Ferritin does not accumulate iron oxidized by caeruloplasmin

1995 ◽  
Vol 305 (1) ◽  
pp. 21-23 ◽  
Author(s):  
A Treffry ◽  
D Gelvan ◽  
A M Konijn ◽  
P M Harrison
Keyword(s):  
Dissolved Oxygen ◽  
Storage Protein ◽  
Iron Oxidation ◽  
Primary Effect ◽  
Significant Extent ◽  
Iron Storage ◽  
Horse Spleen Ferritin ◽  
Iron Storage Protein

Ferritin is an iron-storage protein ubiquitous in mammals, plants and bacteria. It can be reconstituted, in vitro, from the apoprotein and Fe(II) salts in the presence of dissolved oxygen. Recently it has been reported that caeruloplasmin can facilitate apoferritin reconstitution and that iron oxidized by caeruloplasmin is sequestered within the ferritin shell. Here we show that the primary effect of adding caeruloplasmin to horse spleen ferritin during reconstitution is the competition between the two molecules for the iron. This competition results in overall increased rates of iron oxidation and a mixture of products, namely iron-containing ferritin and iron hydroxy polymers attached to caeruloplasmin. Iron oxidized by caeruloplasmin is not incorporated, to any significant extent, into horse spleen ferritin.

Effects of pH on Ca2+i, Na+i, and pHi of MDCK cells: Na(+)-Ca2+ and Na(+)-H+ antiporter interactions

1991 ◽  
Vol 261 (3) ◽  
pp. C482-C489 ◽  
Author(s):  
A. B. Borle ◽  
C. Bender
Keyword(s):  
Mdck Cells ◽  
Extracellular Ph ◽  
Intracellular Acidosis ◽  
Experimental Conditions ◽  
Fluorescent Indicators ◽  
Reverse Mode ◽  
Effects Of Ph ◽  
Ph Stat ◽  
Stat Method ◽  
Free Media

The effects of high and low extracellular pH (8.0 and 6.8) on the intracellular concentration of Ca2+, Na+, and H+ were measured in perfused Madin-Darby canine kidney (MDCK) cells cast in agarose gel threads. Cytosolic free Ca2+ (Ca2+i) was measured with aequorin, while intracellular Na+ (Na+i) and H+ (Hi+ or pHi) were determined with the fluorescent indicators SBFI and BCECF, respectively. In addition, H+ secretion was assayed by the pH-stat method, and Na+ or Ca2+ fluxes were measured with 22Na or 45Ca, respectively. H+ secretion was significantly depressed by several experimental conditions that are known to inhibit the Na(+)-H+ antiporter: H+ secretion decreased 44% in the presence of 10(-5) M ethylisopropylamiloride, 49% in Na+o-free media, 44% in the presence of 10(-4) M ouabain, and 32% in the presence of 10(-4) M 8-bromoadenosine 3',5'-cyclic monophosphate. In addition, pHi decreased by 0.2 pH units in Na+o-free media. Finally, recovery from an intracellular acidosis evoked by 20 mM NH4Cl pulse required the presence of extracellular Na+. When the extracellular pH (pHo) was increased from 7.4 to 8.0, H+ secretion increased 58% from 17.5 to 27.7 nmol.min-1.mg protein-1 and Na+ influx increased 48%. As a result, pHi rose from 7.43 to 7.71 and Na+i increased from 15.6 to 19.7 mM. Finally, Ca2+i rose from 120 to 268 nM. These results suggest that the high pHo stimulated the Na(+)-H+ antiporter, and the subsequent rise in Na+i decreased the Na+ electrochemical potential, thereby activating the reverse mode of Na(+)-Ca2+ exchange (Ca2+ influx vs. Na+ efflux) which led to the rise in Ca2+i.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Evidence that the specificity of iron incorporation into homopolymers of human ferritin L- and H-chains is conferred by the nucleation and ferroxidase centres

1996 ◽  
Vol 314 (1) ◽  
pp. 139-144 ◽  
Author(s):  
Paolo SANTAMBROGIO ◽  
Sonia LEVI ◽  
Anna COZZI ◽  
Barbara CORSI ◽  
Paolo AROSIO
Keyword(s):  
Storage Proteins ◽  
Iron Storage ◽  
Cellular Iron ◽  
Iron Incorporation ◽  
Ferroxidase Activity ◽  
Iron Storage Proteins ◽  
The Difference ◽  
Induced Aggregation

Mammalian ferritins are iron-storage proteins made of 24 subunits of two types: the H- and L-chains. L-chains, in contrast with H-chains, lack detectable ferroxidase activity. When ferritins were subjected to iron loading in vitro with increments near the saturation limit of 4000 Fe atoms per molecule, the homopolymers of human H-chains formed insoluble aggregates, caused by non-specific iron hydrolysis, whereas the homopolymers of L-chains remained soluble and incorporated most of the available iron. To analyse the molecular reasons for the difference, Glu-57 and Glu-60, which are conserved and exposed on the cavity of L-chains, were substituted with His, as in H-chains. The double substitution made the L-homopolymers as sensitive as the H-homopolymers to the iron-induced aggregation, whereas the opposite substitution in the H-chain increased homopolymer resistance to the aggregation only marginally. Millimolar concentrations of citrate and phosphate increased iron incorporation in H-homopolymers by reducing non-specific iron hydrolysis, but inhibited that in L-homopolymers by sequestering available iron. The data indicate that the specific iron incorporation into L-homopolymers is mainly due to the iron-nucleation capacity of Glu-57, Glu-60 and other carboxyl groups exposed on the cavity; in contrast, the specificity of iron incorporation into H-homopolymers is related to its ferroxidase activity, which determines rapid Fe(III) accumulation inside the cavity. The finding that ferroxidase centres are essential for the incorporation of iron in the presence of likely candidates of cellular iron transport, such as phosphate and citrate, confirms their importance in ferritin function in vivo.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

Observations of Frozen-Hydrated Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 504-505
Author(s):  
D. Chrétien ◽  
D. Job ◽  
R.H. Wade
Keyword(s):  
Fourier Transforms ◽  
Structural Complexity ◽  
Image Contrast ◽  
Phase Behaviour ◽  
Experimental Conditions ◽  
Thin Sections ◽  
Surface Lattice ◽  
Cilia And Flagella ◽  
Outstanding Question

Microtubules are filamentary structures found in the cytoplasm of eukaryotic cells, where, together with actin and intermediate filaments, they form the components of the cytoskeleton. They have many functions and show various levels of structural complexity as witnessed by the singlet, doublet and triplet structures involved in the architecture of centrioles, basal bodies, cilia and flagella. The accepted microtubule model consists of a 25 nm diameter hollow tube with a wall made up of 13 paraxial protofilaments (pf). Each pf is a string of aligned tubulin dimers. Some results have suggested that the pfs follow a superhelix. To understand how microtubules function in the cell an accurate model of the surface lattice is one of the requirements. For example the 9x2 architecture of the axoneme will depend on the organisation of its component microtubules. We should also note that microtubules with different numbers of pfs have been observed in thin sections of cellular and of in-vitro material. An outstanding question is how does the surface lattice adjust to these different pf numbers?We have been using cryo-electron microscopy of frozen-hydrated samples to study in-vitro assembled microtubules. The experimental conditions are described in detail in this reference. The results obtained in conjunction with thin sections of similar specimens and with axoneme outer doublet fragments have already allowed us to characterise the image contrast of 13, 14 and 15 pf microtubules on the basis of the measured image widths, of the the image contrast symmetry and of the amplitude and phase behaviour along the equator in the computed Fourier transforms. The contrast variations along individual microtubule images can be interpreted in terms of the geometry of the microtubule surface lattice. We can extend these results and make some reasonable predictions about the probable surface lattices in the case of other pf numbers, see Table 1. Figure 1 shows observed images with which these predictions can be compared.

Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

1981 ◽  
Vol 45 (03) ◽  
pp. 290-293 ◽  
Author(s):  
Peter H Levine ◽  
Danielle G Sladdin ◽  
Norman I Krinsky
Keyword(s):  
Superoxide Dismutase ◽  
Cytochrome C ◽  
Xanthine Oxidase ◽  
Direct Effect ◽  
Platelet Function ◽  
Experimental Conditions ◽  
Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

A Reinvestigation of the Deceptively Simple Reaction of Toluene with ●OH, and the Fate of the Benzyl Radical. I. a Combined Thermodynamic and Kinetic Study on the Competition Between ●OH Addition and Hydrogen Abstraction Reactions.

2019 ◽  
Author(s):  
Zoi Salta ◽  
Agnie M. Kosmas ◽  
Marc E. Segovia ◽  
Martina Kieninger ◽  
Oscar Ventura ◽  
...  
Keyword(s):  
Density Functional ◽  
Room Temperature ◽  
Hydrogen Abstraction ◽  
Reaction Rates ◽  
Composite Model ◽  
Alternative Mechanism ◽  
Radical Intermediate ◽  
Experimental Conditions ◽  
Methyl Group ◽  
Benzyl Radical

This work reports density functional and composite model chemistry calculations performed on the reactions of toluene with the hydroxyl radical. Both experimentally observed H-abstraction from the methyl group and possible additions to the phenyl ring were investigated. Reaction enthalpies and heights of the barriers suggest that H-abstraction is more favorable than ●OH addition to the ring. The calculated reaction rates at room temperature and the radical-intermediate product fractions support this view. This is somehow contradictory with the fact that, under most experimental conditions, cresols are observed in a larger concentration than benzaldehyde. Since the accepted mechanism for benzaldehyde formation involves H-abstraction, a contradiction arises that begs for an explanation. In this first part of our work we give the evidences that support the preference of hydrogen abstraction over ●OH addition and suggest an alternative mechanism which shows that cresols can actually arise also from the former reaction and not only from the latter.

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