Hypothermia for myocardial protection or storage of vascular grafts may damage the endothelium and impair vascular function upon reperfusion/rewarming. Catalytic iron pools and oxidative stress are important mediators of cold-induced endothelial injury. Because endothelial cells are highly adaptive, we hypothesized that hypothermic preconditioning (HPC) protects cells at 0 degrees C by a heme oxygenase-1 (HO-1) and ferritin-dependent mechanism. Storage of human coronary artery endothelial cells at 0 degrees C caused the release of lactate dehydrogenase, increases in bleomycin-detectible iron (BDI), and increases in the ratio of oxidized/reduced glutathione, signifying oxidative stress. Hypoxia increased injury at 0 degrees C but did not increase BDI or oxidative stress further. HPC at 25 degrees C for 15-72 h attenuated these changes by an amount achievable by pretreating cells with 10-20 muM deferoxamine, an iron chelator, and protected cell viability. Treating cells with hemin chloride at 37 degrees C transiently increased intracellular heme, HO-1, BDI, and ferritin. Elevated heme/iron sensitized cells to 0 degrees C but ferritin was protective. HPC increased iron maximally after 2 h at 25 degrees C and ferritin levels peaked after 15 h. HO-1 was not induced. When HPC-mediated increases in ferritin were blocked by deferoxamine, protection at 0 degrees C was diminished. We conclude that HPC-mediated endothelial protection from hypothermic injury is an iron- and ferritin-dependent process.

Purified Trematomus bernacchii bile IgM analysed by SDS-PAGE under reducing and non-reducing conditions consisted essentially of tetramers of the basic structure H2L2. The relative molecular mass of the glycosilated H chain was 76 kDa, while that of L chain was 25 kDa. In addition, the presence in the liver of IgM and mu chain-specific mRNA was demonstrated. Immunohistochemistry detected IgH- and IgL-reactivity in perisinusoidal cells, bile canaliculi and pre-ductules. In the anterior intestine, the intraluminal mucus retained a significant Ig-immunoreactivity, while the mucosa housed a limited density of Ig-producing cells. These findings strongly indicate that Ig could be transported across the hepatocytes to be secreted into the bile and protect the intestinal epithelium. In addition, extravasated plasma cells accumulated within liver portal tracts and close to the capsule that, in turn, was evenly coated by Ig molecules at the peritoneal surface.

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.

The stability of the dodecameric Listeria monocytogenes Dps has been compared with that of the Listeria innocua protein. The two proteins differ only in two amino acid residues that form an intersubunit salt-bridge in L. innocua Dps. This salt-bridge is replaced by a hydrogen bonding network in L. monocytogenes Dps as revealed by the X-ray crystal structure. The resistance to low pH and high temperature was assayed for both Dps proteins under equilibrium conditions and kinetically. Despite the identical equilibrium behavior, significant differences in the kinetic stability and activation energy of the unfolding process are apparent at pH 1.5. The higher stability of L. monocytogenes Dps has been accounted for in terms of the persistence of the hydrogen bonding network at this low pH value. In contrast, the salt-bridge between Lys 114 and Asp 126 characteristic of L. innocua Dps is most likely abolished due to protonation of Asp 126. Proteins 2007.(c) 2006 Wiley-Liss, Inc.

Listeria innocua Dps (DNA binding protein from starved cells) affords protection to DNA against oxidative damage and can accumulate about 500 iron atoms within its central cavity through a process facilitated by a ferroxidase center. The chemistry of iron binding and oxidation in Listeria Dps (LiDps, formerly described as a ferritin) using H(2)O(2) as oxidant was studied to further define the mechanism of iron deposition inside the protein and the role of LiDps in protecting DNA from oxidative damage. The relatively strong binding of 12 Fe(2+) to the apoprotein (K(D) approximately 0.023 microM) was demonstrated by isothermal titration calorimetry, fluorescence quenching, and pH stat experiments. Hydrogen peroxide was found to be a more efficient oxidant for the protein-bound Fe(2+) than O(2). Iron(II) oxidation by H(2)O(2) occurs with a stoichiometry of 2 Fe(2+)/H(2)O(2) in both the protein-based ferroxidation and subsequent mineralization reactions, indicating complete reduction of H(2)O(2) to H(2)O. Electron paramagnetic resonance (EPR) spin-trapping experiments demonstrated that LiDps attenuates the production of hydroxyl radical by Fenton chemistry. DNA cleavage assays showed that the protein, while not binding to DNA itself, protects it against the deleterious combination of Fe(2+) and H(2)O(2). The overall process of iron deposition and detoxification by LiDps is described by the following equations. For ferroxidation, Fe(2+)+ Dps(Z)-->[(Fe(2+))-Dps](Z+1)+ H(+)(Fe(2+) binding) and [(Fe(2+))-Dps](Z+1)+ Fe(2+)+ H(2)O(2)-->[(Fe(3+))(2)(O)(2)-Dps](Z+1)+ 2H(+)(Fe(2+) oxidation/hydrolysis). For mineralization, 2Fe(2+)+ H(2)O(2)+ 2H(2)O --> 2Fe(O)OH((core))+ 4H(+)(Fe(2+) oxidation/hydrolysis). These reactions occur in place of undesirable odd-electron redox processes that produce hydroxyl radical.

The Gram-positive bacterium Listeria innocua possesses an authentic ferritin with an unusual dodecameric assemblage that resembles the quaternary structure of the DNA-binding proteins designated Dps (DNA-binding proteins from starved cells). The L. innocua gene encoding the above protein, termed ferritin from Listeria innocua (fri), has been localized on a 3-kb HindIII chromosomal fragment cloned in the Escherichia coli strain DH5alphaF'. DNA sequence analysis reveals an open reading frame of 468 nucleotides matching perfectly the amino acid sequence of the protein. Primer extension analysis indicates the presence of two transcriptional startpoints located 36 (proximal) and 85 nt (distal) upstream the fri start codon, respectively. Each transcriptional startpoint is preceded by suitably located -10 and -35 elements, which match the sigma(A)(proximal) and sigma(B)(distal) consensus sequences.In L. innocua and Liseria monocytogenes, fri expression increases both upon entry into stationary phase and, more markedly, under low-iron growth conditions. The effect of iron is apparent in the exponential and stationary phases of growth. An up-regulation by iron limitation has never been observed in other proven ferritins and bacterioferritins, but has been reported for several members of the Dps family. The unusual regulation by iron of the Listeria ferritin gene provides further support to the evolutionary link with the Dps family and suggests that the iron storage function may not be the unique role of ferritin in the physiology of this bacterium.

The multi-domain enzyme isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix was studied by denaturant-induced unfolding. At pH 7.5, changes in circular dichroism ellipticity and intrinsic fluorescence showed a complex unfolding transition, whereas at pH 3.0, an apparently two-state and highly reversible unfolding occurred. Analytical ultracentrifugation revealed the dissociation from dimer to monomer at pH 3.0. The thermodynamic and kinetic stability were studied at pH 3.0 to explore the role of inter-domain interactions independently of inter-subunit interplay on the wild type and R211M, a mutant where a seven-membered inter-domain ionic network has been disrupted. The unfolding and folding transitions occurred at slightly different denaturant concentrations even after prolonged equilibration time. The difference between the folding and the unfolding profiles was decreased in the mutant R211M. The apparent Gibbs free energy decreased approximately 2 kcal/mol and the unfolding rate increased 4.3-fold in the mutant protein, corresponding to a decrease in activation free energy of unfolding of 0.86 kcal/mol. These results suggest that the inter-domain ionic network might be responsible for additional stabilization through a significant kinetic barrier in the unfolding pathway that could also explain the larger difference observed between the folding and unfolding transitions of the wild type.

According to their fold, pyridoxal 5'-phosphate-dependent enzymes are grouped into five superfamilies. Fold Type I easily comprises the largest and most investigated group. The enzymes of this group have very similar 3D structures. Remarkably, the location of the cofactor in the active site, between the two domains that form a single subunit, is almost identical in all members of the group. Nonetheless, Fold Type I enzymes show very little sequence identity, raising the question as to which structural features determine the common fold. An important fold determinant appears to be the presence of three evolutionarily conserved clusters of hydrophobic contacts. A previous investigation, which used Escherichia coli serine hydroxymethyltransferase, a well characterized Fold Type I member, demonstrated the involvement of one of these clusters in the stability of the quaternary structure. The present study focuses on the role of the same cluster in the stability of the cofactor binding site. The investigation was carried out by equilibrium denaturation experiments on serine hydroxymethyltransferase forms in which the hydrophobic contact area of the cluster under study was reduced by site-directed mutagenesis. The results obtained show that the mutations clearly affected the process of pyridoxal 5'-phosphate dissociation induced by urea, reducing the stability of the cofactor binding site. We suggest that the third cluster promotes the formation of a bridging structural region that stabilizes the overall protein structure by connecting the two domains, shaping the cofactor binding site and participating in the formation of the quaternary structure. Structured digital abstract * MINT-7293394, MINT-7293405, MINT-7293418: eSHMT (uniprotkb:P0A825) and eSHMT (uniprotkb:P0A825) bind (MI:0407) by cosedimentation in solution (MI:0028).

Dps proteins (DNA binding proteins from starved cells) belong to a widespread bacterial family expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton mediated oxidative stress as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radicals production. The present work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role of the Dps protein cage.

Elucidating pore function at the three-fold channels of 12-subunit, microbial Dps proteins is important in understanding their role in the management of iron/hydrogen peroxide. The Dps pores are called "ferritin-like" because of the structural resemblance to the three-fold channels of 24-subunit ferritins used for iron entry and exit to and from the protein cage. In ferritins, negatively charged residues lining the pores generate a negative electrostatic gradient that guides iron ions towards the ferroxidase centers for catalysis with oxidant and destined for the mineralization cavity. To establish whether the set of three aspartate residues that line the pores in L. innocua Dps act in a similar fashion, D121N, D126N, D130N and D121N-D126N-D130N proteins were produced; kinetics of iron uptake/release and the size distribution of the iron mineral in the protein cavity were compared. The results, discussed in the framework of crystal growth in a confined space, indicate that iron uses the hydrophilic three-fold pores to traverse the protein shell. For the first time, the strength of the electrostatic potential is observed to modulate kinetic cooperativity in the iron uptake/release processes and accordingly the size distribution of the microcrystalline iron minerals in the Dps protein population.

Bacterial and archaeal endo-beta-1,3-glucanases that belong to glycoside hydrolase family 16 share a beta-jelly-roll fold, but differ significantly in sequence and in substrate specificity. The crystal structure of the laminarinase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus (pfLamA) has been determined at 2.1 A resolution by molecular replacement. The pfLamA structure reveals a kink of six residues (72-77) at the entrance of the catalytic cleft. This peptide is absent in the endoglucanases from alkaliphilic Nocardiopsis sp. strain F96 and Bacillus macerans, two proteins displaying an overall fold similar to that of pfLamA, but with different substrate specificity. A deletion mutant of pfLamA, lacking residues 72-75, hydrolyses the mixed-linkage beta-1,3-1,4-glucan lichenan 10 times more efficiently than the wild-type protein, indicating the importance of the kink in substrate preference.

Pyridoxal 5'-phosphate-dependent enzymes may be grouped into five structural superfamilies of proteins, corresponding to as many fold types. The fold type I is by far the largest and most investigated group. An important feature of this fold, which is characterized by the presence of two domains, appears to be the existence of three clusters of evolutionarily conserved hydrophobic contacts. Although two of these clusters are located in the central cores of the domains and presumably stabilize their scaffold, allowing the correct alignment of the residues involved in cofactor and substrate binding, the role of the third cluster is much less evident. A site-directed mutagenesis approach was used to carry out a model study on the importance of the third cluster in the structure of a well characterized member of the fold type I group, serine hydroxymethyltransferase from Escherichia coli. The experimental results obtained indicated that the cluster plays a crucial role in the stabilization of the quaternary, native assembly of the enzyme, although it is not located at the subunit interface. The analysis of the crystal structure of serine hydromethyltransferase suggested that this stabilizing effect may be due to the strict structural relation between the cluster and two polypeptide loops, which, in fold type I enzymes, mediate the interactions between the subunits and are involved in cofactor binding, substrate binding and catalysis.

On the basis of available data with regard to the chemical and physical properties of the "substrate" luciferin (LH(2)) and enzyme, luciferase (A), and of kinetic data derived both from the reaction in extracts of Cypridina, and from the luminescence of intact bacteria, the fundamental reactions involved in the phenomenon of bioluminescence have been schematized. These reactions provide a satisfactory basis for interpreting the known characteristics of the system, as well as the theoretical chemistry with regard to the control of its over-all velocity in relation to various factors. These factors, here studied experimentally wholly with bacteria, Photobacterium phosphoreum in particular, include pH, temperature, pressure, and the drugs sulfanilamide, urethane, and alcohol, separately and in relation to each other. Under steady state conditions of bacterial luminescence, with excess of oxidizable substrate and with oxygen not limiting, the data indicate that the chief effects of these agents center around the pace setting reactions, which may be designated by the equation: A + LH(2)--> ALH(2) following which light emission is assumed proportional to the amount of the excited molecule, AL*. The relation between pH and luminescence intensity varies with (a), the buffer mixture and concentration,(b), the temperature, and (c), the hydrostatic pressure. At an optimum temperature for luminescence of about 22 degrees C. in P. phosphoreum, the effects of increasing or decreasing the hydrogen ion concentration are largely reversible over the range between pH 3.6 and pH 8.8. The relation between luminescence intensity and pH, under the experimental conditions employed, is given by the following equation, in which I(1) represents the maximum intensity, occurring about pH 6.5; I(2) the intensity at any other given pH; K(5) the equilibrium constant between hydrogen ions and the AL(-); and K(6) the corresponding constant with respect to hydroxyl ions: See PDF for Equation The value of K(5), as indicated by the data, amounts to 4.84 x 10(4), while that of K(6) amounts to 4.8 x 10(5). Beyond the range between approximately pH 3.8 and 8.8, destructive effects of the hydrogen and hydroxyl ions, respectively, were increasingly apparent. By raising the temperature above the optimum, the destructive effects were apparent at all pH, and the intensity of the luminescence diminished logarithmically with time. With respect to pH, the rate of destruction of the light-emitting system at temperatures above the optimum was slowest between pH 6.5 and 7.0, and increased rapidly with more acid or more alkaline reactions of the medium. The reversible effects of slightly acid pH vary with the temperature in the manner of an inhibitor (Type I) that acts independently of the normal, reversible denaturation equilibrium (K(1)) of the enzyme. The per cent inhibition caused by a given acid pH in relation to the luminescence intensity at optimum pH, is much greater at low temperatures, and decreases as the temperature is raised towards the optimum temperature. The observed maximum intensity of luminescence is thus shifted to slightly higher temperatures by increase in (H(+)). The apparent activation energy of luminescence is increased by a decrease in pH. The value of DeltaHdouble dagger at pH 5.05 was calculated to be 40,900 calories, in comparison with 20,700 at a pH of 6.92. The difference of 20,200 is taken to represent an estimate of the heat of ionization of ALH in the activation process, and compares roughtly with the 14,000 calories estimated for the same process, by analyzing the data from the point of view of hydrogen ions as an inhibitor. The decreasing temperature coefficient for luminescence in proceeding from low temperatures towards the optimum is accounted for in part by the greater degree of ionization of ALH. At the optimum temperature and acid reactions, pressures up to about 500 atmospheres retard the velocity of the luminescent oxidation. At the same temperature, with decrease in hydrogen ion concentration, the pressure effect is much less, indicating a considerable volume increase in the process of ionization and activation. In the extremely alkaline range, beyond pH 9, luminescence is greatly reduced, as compared with the intensity at neutrality, and under these conditions pressure causes a pronounced increase in intensity, presumably by acting upon the reversible denaturation equilibrium of the protein enzyme, A. Sulfanilamide, in neutral solutions, acts on luminescence in a manner very much resembling that of hydrogen ions at acidities between pH 4.0 and pH 6.5. Like the hydrogen ion equilibrium, the sulfanilamide equilibrium involves a ratio of approximately one inhibitor molecule to one enzyme molecule. The heat of reaction amounts to about 11,600 calories or more in a reversible combination that evidently evolves heat. Like the action of H ions, sulfanilamide causes a slight shifting of maximum luminescence intensity in the direction of higher temperatures, and an increase in the energy of activation. The effect of sulfanilamide on the growth of broth cultures of eight species of luminous bacteria indicates that there is no regular relationship among the different organisms between the concentration of the drug that prevents growth, and that which prevents luminescence in the cells which develop in the presence of sulfanilamide. p-Aminobenzoic acid (PAB) antagonizes the sulfanilamide inhibition of growth in luminous bacteria, and the cultures that develop are luminous. When (PAB) is added to cells from fully developed cultures, it has no effect on luminescence, or causes a slight inhibition, depending on the concentration. With luminescence partly inhibited by sulfanilamide, the addition of PAB has no effect, or has an inhibitory effect which adds to that caused by sulfanilamide. Two different, though possibly related, enzyme systems thus appear to limit growth and luminescence, respectively. The possible mechanism through which both the inhibitions and the antagonism take place is discussed. The irreversible destruction of the luminescent system at temperatures above that of the maximum luminescence, in a medium of favorable pH to which no inhibitors have been added, proceeds logarithmically with time at both normal and increased hydrostatic pressures. Pressure retards the rate of the destruction, and the analysis of the data indicates that a volume increase of roughly 71 cc. per gm. molecule at 32 degrees C. takes place in going from the normal to the activated state in this reaction. At normal pressure, the rate of destruction has a temperature coefficient of approximately 90,000 calories, or about 20,000 calories more than the heat of reaction in the reversible denaturation equilibrium. The data indicate that the equilibrium and the rate process are two distinct reactions. The equation for luminescence intensity, taking into account both the reversible and irreversible phases of the reaction is given below. In the equation b is a proportionality constant; k' the rate constant of the luminescent reaction; A(0) the total luciferase; A(0i) the total initial luciferase at time t equals 0; k(n) the rate constant for the destruction of the native, active form of the enzyme; k(d) the rate constant for the destruction of the reversibly denatured, inactive form; t the time; and the other symbols are as indicated above: See PDF for Equation For reasons cited in the text, k(n) evidently equals k(d). Urethane and alcohol, respectively, act in a manner (Type II) that promotes the breaking of the type of bonds broken in both the reversible and irreversible reactions and so promotes the irreversible denaturation. This result is in contrast to the effects of sulfanilamide, which at appropriate concentrations may give rise to the same initial inhibition as that caused by urethane, but remains constant with time. The inhibition caused by urethane and alcohol, respectively, increases as the temperature is raised. As a result, the apparent optimum is shifted to lower temperatures, and the activation energy for the over-all process of luminescence diminishes. An analysis for the approximate heat of reaction in the equilibrium between these drugs and the enzyme, indicates 65,000 calories for urethane, and 37,000 for alcohol. A similar analysis with respect to the effect of hydroxyl ions as the inhibitor gives 60,300 calories. The effects of alcohol and urethane are sensitive to hydrostatic pressure. Moderate inhibitions at optimum temperature and pH, caused by relatively small concentrations of either drug, are completely abolished by pressures of 3,000 to 4,000 pounds per square inch. At optimum temperature and pH, increasing concentrations of alcohol caused the apparent optimum pressure for luminescence to shift markedly in the direction of higher pressures. Analysis of the data with respect to concentration of alcohol at different pressures indicated that the ratio of alcohol to enzyme molecules amounted to approximately 4, at 7,000 pounds, but only about 2.8 at normal pressures. This phenomenon was taken to indicate that more than one equilibrium is established between the alcohol and the protein. A similar interpretation was suggested in connection with the fact that analysis of the relation between concentration of urethane and amount of inhibition at different temperatures also indicated a ratio of urethane to enzyme molecules that increased with temperature in the equilibria involved. Analysis of the data with respect to pressure and the inhibition caused by a given concentration of alcohol at different temperatures indicated that the volume change involved in the combination of alcohol with the enzyme must be very small, while the actual effect of pressure is apparently mediated through the reversible denaturation of the protein enzyme, which is promoted by alcohol, urethane, and drugs of similar type.

A novel ferritin cDNA, SferH-5, has been cloned from 7-day-old soybean seedlings. Putative SferH-5 has 96% identity with SferH-1 reported previously. All the five amino acid variants distributed in the mature region are not involved in highly conserved residues associated with ferroxidase activity center. We speculate that SferH-5 encodes a novel 26.5-kDa subunit of soybean seed ferritin, which is designated H-5 in this study. Recombinant H-5 was able to assemble, together with co-expressed H-2, as a functional soybean seed ferritin-like complex, H-5/H-2. Our data reveal the potential heterogeneity of the 26.5-kDa subunit of soybean seed ferritin.

The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of - 1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.

Levels of ubiquitin (Ub)-conjugated proteins, as an index of misfolded or damaged proteins, were measured in notothenioid fishes, with both Antarctic (Trematomus bernacchii, T. pennellii, Pagothenia borchgrevinki) and non-Antarctic (Notothenia angustata, Bovichtus variegatus) distributions, as well as non-notothenioid fish from the Antarctic (Lycodichthys dearborni, Family Zoarcidae) and New Zealand (Bellapiscis medius, Family Tripterygiidae), in an effort to better understand the effect that inhabiting a sub-zero environment has on maintaining the integrity of the cellular protein pool. Overall, levels of Ub-conjugated proteins in cold-adapted Antarctic fishes were significantly higher than New Zealand fishes in gill, liver, heart and spleen tissues suggesting that life at sub-zero temperatures impacts protein homeostasis. The highest tissue levels of ubiquitinated proteins were found in the spleen of all fish. Ub conjugate levels in the New Zealand N. angustata, more closely resembled levels measured in other Antarctic fishes than levels measured in other New Zealand species, likely reflecting their recent shared ancestry with Antarctic notothenioids.

We characterized chicken erythrocyte and human platelet ferritin by biochemical studies and immunofluorescence. Erythrocyte ferritin was found to be a homopolymer of H-ferritin subunits, resistant to proteinase K digestion, heat stable, and contained iron. In mature chicken erythrocytes and human platelets, ferritin was localized at the marginal band, a ring-shaped peripheral microtubule bundle, and displayed properties of bona fide microtubule-associated proteins such as tau. Red blood cell ferritin association with the marginal band was confirmed by temperature-induced disassembly-reassembly of microtubules. During erythrocyte differentiation, ferritin co-localized with coalescing microtubules during marginal band formation. In addition, ferritin was found in the nuclei of mature erythrocytes, but was not detectable in those of bone marrow erythrocyte precursors. These results suggest that ferritin has a function in marginal band formation and possibly in protection of the marginal band from damaging effects of reactive oxygen species by sequestering iron in the mature erythrocyte. Moreover, our data suggest that ferritin and syncolin, a previously identified erythrocyte microtubule-associated protein, are identical. Nuclear ferritin might contribute to transcriptional silencing or, alternatively, constitute a ferritin reservoir.

In the present study, we describe the purification and molecular characterization of Cu,Zn superoxide dismutase (SOD) from Trematomus bernacchii, a teleost widely distributed in many areas of Antarctica, that plays a pivotal role in the Antarctic food chain. The amino acid and cDNA sequences have been obtained using both biochemical and molecular biology approaches and are compared with Cu,Zn SODs from other fishes. Assessment of the primary sequences highlights that the catalytically important residues are fully conserved in Cu,Zn SOD from T. bernacchii. Phylogenetic analyses performed on Cu,Zn SOD amino acid sequences permit speculation regarding the evolution of this protein. In particular, the data confirms the erratic differentiation of these proteins and concurs with the theory of the "unclock-like" behaviour of Cu,Zn SOD evolution.

Ferritin, the iron-storing protein of mammals, is known to darken T(2)-weighted magnetic resonance images. This darkening can be used to noninvasively measure an organ's iron content. Significant discrepancies exist between T(2) data obtained with ferritin-containing tissues and with aqueous solutions of horse spleen ferritin (HSF). The NMR properties of stable human ferritin have never been studied in aqueous solutions. Relaxometry results on human liver and spleen ferritin are reported here, showing that the relaxation induced in aqueous solutions by human ferritins is comparable to that induced by HSF. As a consequence, the differences between ferritin-containing human tissues and ferritin solutions cannot be attributed to different NMR properties of human and horse ferritins, but probably to a clustering of the protein in vivo.

The Dps family members constitute a distinct group of multimeric and ferritin-like iron binding proteins (up to 500 iron atoms/12-mer) that are widespread in eubacteria and archaea and implicated in oxidative stress resistance and virulence. Despite the wealth of structural knowledge, the mechanism of iron incorporation has remained elusive. Here, we provide evidence on Dpr of the swine and human pathogen Streptococcus suis that:(i) iron incorporation proceeds by Fe(II) binding, Fe(II) oxidation and subsequent storage as Fe(III);(ii) Fe(II) atoms enter the 12-mer cavity through four hydrophilic pores; and (iii) Fe(II) atoms are oxidized inside the 12-mer cavity at 12 identical inter-subunit sites, which are structurally different but functionally equivalent to the ferroxidase centres of classical ferritins. We also provide evidence, by deleting and ectopically overexpressing Dpr, that Dpr affects cellular iron homeostasis. The key residues responsible for iron incorporation in S. suis Dpr are well conserved throughout the Dps family. A model for the iron incorporation mechanism of the Dps/Dpr ferritin-like protein is proposed.

Canine and equine ferritin H and L subunit cDNA clones were obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) and TA cloning from various tissues. Canine liver and spleen ferritin H subunit cDNA clones contained an open reading frame for the same 182-amino acid protein as that reported in canine brain ferritin H subunit cDNA although there were substitutions in the 3'-noncoding regions. Ferritin L subunit cDNA clones from canine liver, spleen, and kidney showed identical coding sequences encoding the 174-amino acid protein except for a single nucleotide substitution in kidney (C474G). The H subunit nucleotide sequences of equine leukocyte and spleen were identical to the fragment encoding the 181-amino acid protein in equine peripheral blood mononuclear cells, with the exception of one substitution seen in both leukocyte and spleen sequences (C234T). The nucleotide sequence of equine leukocyte ferritin L subunit showed 7 substitutions compared with the published equine liver L subunit sequence with two substitutions at positions 281 and 282 resulting in an amino acid substitution of P94L. The amino acid residues involved in the ferroxidase center and in iron nucleation were perfectly conserved in H and L subunits of canine and equine ferritins, respectively.

To test the temperature sensitivity of molecular chaperones in poikilothermic animals, we purified the molecular chaperone Hsc70 from 2 closely related notothenioid fishes--the Antarctic species Trematomus bernacchii and the temperate New Zealand species Notothenia angustata--and characterized the effect of temperature on Hsc70 adenosine triphosphatase (ATPase) activity. Hsc70 ATPase activity was measured using [alpha-32P]-adenosine triphosphate (ATP)-based in vitro assays followed by separation of adenylates by thin-layer chromatography. For both species, a significant increase in Hsc70 ATPase activity was observed across a range of temperatures that was ecologically relevant for each respective species. Hsc70 from T bernacchii hydrolyzed 2-fold more ATP than did N angustata Hsc70 at 0 degrees C, suggesting that the Antarctic molecular chaperone may be adapted to function more efficiently at extreme cold temperatures. In addition, Q10 measurements indicate differential temperature sensitivity of the ATPase activity of Hsc70 from these differentially adapted fish that correlates with the temperature niche inhabited by each species. Hsc70 from T bernacchii was relatively temperature insensitive, as indicated by Q10 values calculated near 1.0 across each temperature range measured. In the case of Hsc70 purified from N angustata, Q10 values indicated thermal sensitivity across the temperature range of 0 degrees C to 10 degrees C, with a Q10 of 2.714. However, Hsc70 from both T bernacchii and N angustata exhibited unusually high thermal stabilities with ATPase activity at temperatures that far exceeded temperatures encountered by these fish in nature. Overall, as evidenced by in vitro ATP hydrolysis, Hsc70 from T bernacchii and N angustata displayed biochemical characteristics that were supportive of molecular chaperone function at ecologically relevant temperatures.