Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.     

20S proteasome and accumulation of oxidized and ubiquitinated proteins in maize leaves subjected to cadmium stress

In order to examine the possible involvement of the 20S proteasome in degradation of oxidized proteins, the effects of different cadmium concentrations on its activities, protein abundance and oxidation level were studied using maize (Zea mays L.) leaf segments. The accumulation of carbonylated and ubiquitinated proteins was also investigated. Treatment with 50 microM CdCl(2) increased both trypsin- and PGPH-like activities of the 20S proteasome. The incremental changes in 20S proteasome activities were probably caused by an increased level of 20S proteasome oxidation, with this being responsible for degradation of the oxidized proteins. When leaf segments were treated with 100 microM CdCl(2), the chymotrysin- and trypsin-like activities of the 20S proteasome also decreased, with a concomitant increase in accumulation of carbonylated and ubiquitinated proteins. With both Cd(2+) concentrations, the abundance of the 20S proteasome protein remained similar to the control experiments. These results provide evidence for the involvement of this proteolytic system in cadmium-stressed plants.     

Low-temperature magnetic circular dichroism spectra and magnetisation curves of 4Fe clusters in iron-sulphur proteins from Chromatium and Clostridium pasteurianum

The magnetic circular dichroism (MCD) spectra of the 4Fe clusters in the iron-sulphur proteins high-potential iron protein from Chromatium and the 8Fe ferredoxin from Clostridium pasteurianum have been measured over the wavelength range 300-800 nm at temperatures between approx. 1.5 and 50 K and at magnetic fields up to 5 tesla. In both cases the proteins have been studied in the oxidized and reduced states. The reduced state of high-potential iron protein gives a temperature-independent MCD spectrum up to 20 K, confirming the diamagetism of this state at low temperature. The MCD spectrum of samples of oxidized ferredoxin invariably show the presence of a low concentration of a paramagnetic species, in agreement with the observation that the EPR spectrum always shows a signal at g = 2.01. The paramagnetic MCD spectrum runs across the whole of the wavelength range studied and therefore most probably originates from an iron-sulphur centre. The diamagnetic component of the MCD spectrum of oxidized ferredoxin is very similar to that of reduced high-potential iron protein. The low-temperature MCD spectra of oxidized high-potential iron protein and reduced ferredoxin reveal intense, temperature-dependent bands. The spectra are highly structured with that of high-potential iron protein showing a large number of electronic transitions across the visible region. The MCD spectra of the two different oxidation levels are quite distinctive and should provide a means of establishing the identity of these state of 4Fe clusters in more complex proteins. MCD magnetisation curves have been constructed from detailed studies of the field and temperature dependence of the MCD spectra of the two paramagnetic oxidation states. These plots can be satisfactorily fitted to the theoretically computed curves for an S = 1/2 ground state with the g factors experimentally determined by EPR spectroscopy. The low-temperature MCD spectra of the reduced 2Fe-2S ferredoxin from Spirulina maxima are also presented and MCD magnetisation curves plotted and fitted to the experimentally determined g factors.     

The Role of Calpain and Proteasomes in the Degradation of Carbonylated Neuronal Cytoskeletal Proteins in Acute Experimental Autoimmune Encephalomyelitis

The present study was designed to investigate the role of calpain and the proteasome in the removal of oxidized neuronal cytoskeletal proteins in myelin basic protein-induced experimental autoimmune encephalomyelitis (EAE). To this end, EAE rats received a single intrathecal injection of calpeptin or epoxomicin at the first sign of clinical disease. Forty-eight hours later, animals were sacrificed and lumbar spinal cord segments were dissected and used for biochemical analyses. The results show that calpain and proteasome activity is specifically, but partially, inhibited with calpeptin and epoxomicin, respectively. Calpain inhibition causes an increase in total protein carbonylation and in the amount of neurofilament proteins (NFPs), β-tubulin and β-actin that were spared from degradation, but no changes are seen in the oxidation of any of three NFPs. By contrast, proteasome inhibition has no effect on total protein carbonylation or cytoskeletal protein degradation but increases the amount of oxidized NFH and NFM. These results suggest that while the proteasome may contribute to removal of oxidized NFPs, calpain is the main protease involved in degradation of neuronal cytoskeleton and does not preferentially targets oxidized NFPs species in acute EAE. Different results were obtained in a cell-free system, where calpain inhibition rises the amount of oxidized NFH, and proteasome inhibition fails to change the oxidation state of the NFPs. The later finding suggests that the preferential degradation of oxidized NFH and NFM in vivo by the proteasome occurs via the 26S and not the 20S particle.     

Degradation of oxidized proteins by the 20S proteasome

Oxidatively modified proteins are continuously produced in cells by reactive oxygen and nitrogen species generated as a consequence of aerobic metabolism. During periods of oxidative stress, protein oxidation is significantly increased and may become a threat to cell survival. In eucaryotic cells the proteasome has been shown (by purification of enzymatic activity, by immunoprecipitation, and by antisense oligonucleotide studies) to selectively recognize and degrade mildly oxidized proteins in the cytosol, nucleus, and endoplasmic reticulum, thus minimizing their cytotoxicity. From in vitro studies it is evident that the 20S proteasome complex actively recognizes and degrades oxidized proteins, but the 26S proteasome, even in the presence of ATP and a reconstituted functional ubiquitinylating system, is not very effective. Furthermore, relatively mild oxidative stress rapidly (but reversibly) inactivates both the ubiquitin activating/conjugating system and 26S proteasome activity in intact cells, but does not affect 20S proteasome activity. Since mild oxidative stress actually increases proteasome-dependent proteolysis (of oxidized protein substrates) the 20S 'core' proteasome complex would appear to be responsible. Finally, new experiments indicate that conditional mutational inactivation of the E1 ubiquitin-activating enzyme does not affect the degradation of oxidized proteins, further strengthening the hypothesis that oxidatively modified proteins are degraded in an ATP-independent, and ubiquitin-independent, manner by the 20S proteasome. More severe oxidative stress causes extensive protein oxidation, directly generating protein fragments, and cross-linked and aggregated proteins, that become progressively resistant to proteolytic digestion. In fact these aggregated, cross-linked, oxidized proteins actually bind to the 20S proteasome and act as irreversible inhibitors. It is proposed that aging, and various degenerative diseases, involve increased oxidative stress (largely from damaged and electron 'leaky' mitochondria), and elevated levels of protein oxidation, cross-linking, and aggregation. Since these products of severe oxidative stress inhibit the 20S proteasome, they cause a vicious cycle of progressively worsening accumulation of cytotoxic protein oxidation products.     

Nitrogenase X: M?ssbauer and EPR studies on reversibly oxidized MoFe protein from Azotobacter vinelandii OP. Nature of the iron centers.

Under anaerobic conditions the molybdenum-iron protein (MoFe protein) from Azotobacter vinelandii can be reversibly oxidized with thionine. Electron paramagnetic resonance studies reveal that the oxidation proceeds in two distinct phases: the MoFe protein can be oxidized by four electrons without loss of the EPR signal from the S = 3/2 cofactor centers. A second oxidation step, involving two electrons, leads to the disappearance of the cofactor EPR signal. In order to correlate the events during the thionine titration with redox reactions involving individual iron centers we have studied the MoFe proteins from A vinelandii and Clostridium pasteurianum with M?ssbauer spectroscopy. Spectra were taken in the temperature range from 1.5 K to 200 K in applied magnetic fields of up to 54 kG. Analysis of the M?ssbauer data allows us to draw three major conclusions: (1) the holoprotein contains 30 +/- 2 iron atoms. (2) Most probably, 12 iron atoms belong to two, apparently identical, iron clusters (labeled M) which we have shown previously to be structural components of the iron and molybdenum containing cofactor of nitrogenase. The M-centers can be stabilized in three distinct oxidation states, MOXe- in equilibrium MNe- in equilibrium MR. The diamagnetic (S = 0) state MOX is attained by oxidation of the native state MN with either thionine or oxygen. MR is observed under nitrogen fixing conditions. (3) The data strongly suggest that 16 iron atoms are associated with four iron centers which we propose to call P-clusters. Each P-cluster contains four spin-coupled iron atoms. In the native protein the P-clusters are in the diamagnetic state PN, yielding the M?ssbauer signature which we have labeled previously 'components D and Fe2+'. Three irons of the D-type and one iron of the Fe2+-type appear to comprise a P-cluster. A one-electron oxidation yields the paramagnetic state POX. Although the state POX is characterized by half-integral electronic spin a peculiar combination of zero-field splitting parameters and spin relaxation renders this state EPR-silent. Spectroscopically, the P-clusters are novel structures; there is, however, evidence that they are closely related to familiar 4Fe-4S centers.     

Amino acid, peptide, and protein hydroperoxides and their decomposition products modify the activity of the 26S proteasome

Proteins are major biological targets for oxidative damage within cells because of their high abundance and rapid rates of reaction with radicals and singlet oxygen. These reactions generate high yields of hydroperoxides. The turnover of both native and modified/damaged proteins is critical for maintaining cell homeostasis, with this occurring via the proteasomal and endosomal-lysosomal systems; the former is of particular importance for intracellular proteins. In this study we have examined whether oxidation products generated on amino acids, peptides, and proteins modulate 26S proteasome activity. We show that oxidation products, and particularly protein hydroperoxides, are efficient inhibitors of the 26S proteasome tryptic and chymotryptic activities, with this depending, at least in part, on the presence of hydroperoxide groups. Removal of these species by reduction significantly reduces proteasome inhibition. This loss of activity is accompanied by a loss of thiol residues, but an absence of radical formation, consistent with molecular, rather than radical, reactions being responsible for proteasome inhibition. Aldehydes also seem to play a role in the inhibition of chymotryptic activity, with this prevented by treatment with NaBH(4), which reduces these groups. Inhibition occurred at hydroperoxide concentrations of ≥1μM for oxidized amino acids and peptides and ≥10μM for oxidized proteins, compared with ca. 100μM for H(2)O(2), indicating that H(2)O(2) is a much less effective inhibitor. These data indicate that the formation of oxidized proteins within cells may modulate cell function by interfering with the turnover of native proteins and the clearance of modified materials.     

The activation of glycogen synthase in hepatocytes from rats with a glycogen storage disorder (gsd/gsd)

A rapid high resolution method of purification of the Trp-containing S100 proteins (S100a, S100a′) and of the S100b protein has been developed. The principle of this method is based on the fact that S100b protein becomes highly hydrophobic upon Zn²⁺ binding, whereas S100a and S100a′ are not affected. On an affinity chromatography of phenyl—Sepharose column, S100b is selectively bound in presence of zinc, whereas the Trp-containing S100 patients are quickly eluted. The S100b protein is further eluted with a buffer containing EDTA.     

Oxidation of biological membranes and its inhibition. Free radical chain oxidation of erythrocyte ghost membranes by oxygen

The oxidation of human and rat erythrocyte ghost membranes by molecular oxygen has been performed in an aqueous suspension at 37 degrees C. A constant rate of oxygen uptake was observed in the presence of radical initiator. alpha-Tocopherol in the membrane suppressed the oxidation and the induction period was clearly observed. alpha-Tocopherol decreased linearly during the induction period and when it was depleted the induction period was over and a rapid oxidation started. The rate of oxidation was proportional to the square root of the rate of initial radical generation. The kinetic chain length, the ratio of the rate of propagation to that of initiation, was long, ranging from 7 to 100. These results indicate that the erythrocyte ghost membranes are oxidized by a free radical chain mechanism by molecular oxygen. Among the fatty acids of membrane lipids, polyunsaturated fatty acids were oxidized exclusively. Proteins as well as polyunsaturated fatty acids were oxidized and the formation of the high- and low-molecular-weight proteins and the decrease of protein bands were observed on gel electrophoresis.     

The Saccharomyces cerevisiae proteome of oxidized protein thiols: contrasted functions for the thioredoxin and glutathione pathways

Protein thiol oxidation subserves important biological functions and constitutes a sequel of reactive oxygen species toxicity. We developed two distinct thiol-labeling approaches to identify oxidized cytoplasmic protein thiols in Saccharomyces cerevisiae. Inone approach, we used N-(6-(biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide to purify oxidized protein thiols, and in the other, we used N-[(14)C]ethylmaleimide to quantify this oxidation. Both approaches showed a large number of the same proteins with oxidized thiols ( approximately 200), 64 of which were identified by mass spectrometry. We show that, irrespective of its mechanism, protein thiol oxidation is dependent upon molecular O(2). We also show that H(2)O(2) does not cause de novo protein thiol oxidation, but rather increases the oxidation state of a select group of proteins. Furthermore, our study reveals contrasted differences in the oxidized proteome of cells upon inactivation of the thioredoxin or GSH pathway suggestive of very distinct thiol redox control functions, assigning an exclusive role for thioredoxin in H(2)O(2) metabolism and the presumed thiol redox buffer function for GSH. Taken together, these results suggest the high selectivity of cytoplasmic protein thiol oxidation.     

Intra- and Interchain Disulfide Bond Generation in S100b Protein

Disulfide-bridged S100b protein formation, aircatalyzed and induced by thiol/disulfide exchange, was studied under various ionic conditions. As native, physiological disulfide-bridged proteins are obtained easily from their reduced counterparts under appropriate redox conditions, this work was performed to determine whether this was the case for disulfide-bridged S100b proteins, reported to have neurite extension activity. In nondenaturating native medium, no disulfide-bridged species could be generated from reduced proteins in any of the ion-induced conformations tested (no ions, Ca2+, Zn2+, or K+) under widely different redox conditions. Only mixed disulfides accumulated, in certain cases. In contrast, intrasubunit monomeric and intersubunit dimeric disulfide-bridged species were readily and efficiently generated under denaturating conditions. A brief characterization of these oxidized species suggested that they differed widely in structure from their reduced counterparts and that they probably did not bind Ca2+. Taken together, these data question the physiological relevance of these disulfide-bridged S100b protein species.     

Simultaneous detection of changes in protein expression and oxidative modification as a function of age and APOE genotype

To better elucidate temporal changes in protein oxidation resulting from aging and the Alzheimer's disease-associated Apolipoprotein E (ApoE), we developed a 2D-DIGE-based method for simultaneously detecting differential expression and carbonyl oxidation of proteins. Specifically, we examined changes in the levels of oxidation and total protein expression in hippocampi from young-adult (25-30 weeks) and old (76-97 weeks) mice transgenic for the human Apolipoprotein E gene (APOE, APOE3, APOE4) isoforms, APOE3 or APOE4. Protein samples were labeled with either a fluorescent aminooxyacetamide (Alexa Fluor 488) to detect carbonyl modifications or with NHS-Cy3 to detect total protein expression. A protein sample used as an internal control was labeled with NHS-Cy5 and run on each gel. DIGE analysis revealed 38 differentially oxidized and 100 differentially expressed protein spots with significantly different levels (P < 0.05). For oxidized proteins, principal component analysis revealed two distinct clusters: one in which oxidation increased with age independent of APOE genotype, and the second in which oxidation was dependent on APOE genotype. For total protein expression, principal component analysis revealed a large overlap between changes with overall aging and between APOE genotypes. The use of a fluorescent tag to label oxidized proteins, in combination with a NHS-Cy3 to label total protein, makes it possible to determine changes in both protein oxidation and protein expression levels in a single experiment. These studies reveal that the expression levels of peroxiredoxin protein family members Prdx2, 3, and 6 are modified by age, APOE genotype, or both.     

Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins.

The biological activity of some proteins is known to be sensitive to oxidative damage caused by a variety of oxidants. The model protein staphylococcal nuclease was used to explore the effect on protein structural stability of oxidizing methionine to the sulfoxide form. These effects were compared with the effects of substituting methionines with isoleucine and leucine, a potential strategy for stabilizing proteins against oxidative damage. Wild-type nuclease and various mutants were oxidized with hydrogen peroxide. Stabilities of both oxidized and unoxidized proteins were determined by guanidine hydrochloride denaturation. Oxidation destabilized the wild-type protein by over 4 kcal/mol. This large loss of stability supports the idea that in some cases loss of biological activity is linked to disruption of the protein native state. Comparison of mutant protein's stability losses upon oxidation showed that methionines 65 and 98 had a much greater destabilizing effect when oxidized than methionines 26 or 32. While substitution of methionine 98 carried as great an energetic penalty as oxidation, substitution at position 65 was less disruptive than oxidation. Thus a simple substitution mutagenesis strategy to protect a protein against oxidative destabilization is practical for some methionine residues.     

Rat Brain S100b Protein: Purification, Characterization, and Ion Binding Properties. A Comparison with Bovine S100b Protein

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea-sodium dodecyl sulfate-polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal-free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCl, rat brain S100b protein is characterized by two high-affinity Ca2+ binding sites with a KD of 2 X 10(-5) M and four lower affinity sites with KD about 10(-4) M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low-affinity calcium binding sites whereas similar Ca2+-induced conformational changes were observed for both proteins. In the presence of 120 mM KCl rat brain S100b protein bound two Zn2+-ions/mol of protein with a KD of 10(-7) M and four other with lower affinity (KD approximately equal to 10(-6) M). The occupancy of the two high-affinity Zn2+ binding sites was responsible for most of the Zn2+-induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox and spectroscopic properties of oxidized MoFe protein from Azotobacter vinelandii

The MoFe protein from Azotobacter vinelandii undergoes a six-electron oxidation by various organic dye oxidants with full retention of initial activity. Reduction of the oxidized protein by S2O42- and by controlled potential electrolysis indicates the presence of two reduction regions at -290 and -480 mV, each requiring three electrons for complete reaction. Control of the oxidation conditions provides a means for preparing two distinct MoFe protein species selectively oxidized by three electrons. Selective reduction of the redox region at -290 mV causes development of the EPR signal associated with fully reduced MoFe protein while reduction at -480 mV produces a change in the visible spectrum but has no effect on the EPR signal intensity. Kinetic differences for reduction of the two redox regions indicate that the cofactor region undergoes a more rapid reaction with reductant than the other metal redox sites.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Impact of Hydrogen Peroxide on the Activity, Structure, and Conformational Stability of the Oxidized Protein Repair Enzyme Methionine Sulfoxide Reductase A

The oxidized protein repair methionine sulfoxide reductase (Msr) system has been implicated in aging, in longevity, and in the protection against oxidative stress. This system is made of two different enzymes (MsrA and MsrB) that catalyze the reduction of the two diastereoisomers S- and R-methionine sulfoxide back to methionine within proteins, respectively. Due to its role in cellular protection against oxidative stress that is believed to originate from its reactive oxygen species scavenging ability in combination with exposed methionine at the surface of proteins, the susceptibility of MsrA to hydrogen-peroxide-mediated oxidative inactivation has been analyzed. This study is particularly relevant to the oxidized protein repair function of MsrA in both fighting against oxidized protein formation and being exposed to oxidative stress situations. The enzymatic properties of MsrA indeed rely on the activation of the catalytic cysteine to the thiolate anion form that is potentially susceptible to oxidation by hydrogen peroxide. The residual activity and the redox status of the catalytic cysteine were monitored before and after treatment. These experiments showed that the enzyme is only inactivated by high doses of hydrogen peroxide. Although no significant structural modification was detected by near- and far-UV circular dichroism, the conformational stability of oxidized MsrA was decreased as compared to that of native MsrA, making it more prone to degradation by the 20S proteasome. Decreased conformational stability of oxidized MsrA may therefore be considered as a key factor for determining its increased susceptibility to degradation by the proteasome, hence avoiding its intracellular accumulation upon oxidative stress.     

Structural and catalytic properties of oxidized and reduced chloroplast NADP-malate dehydrogenase upon denaturation and renaturation

Chloroplast NADP-dependent malate dehydrogenase exists in two interconvertible forms: the inactive disulfide-containing form and the active dithiol form. No major difference in secondary structure or conformation was found between the oxidized and the reduced enzyme as determined by circular dichroism and intrinsic protein fluorescence. The guanidine/HCl-dependent unfolding of the enzyme is characterized by two transition midpoints: those of the reduced enzyme are lower by about 0.2 M guanidine/HCl compared to the oxidized enzyme. As shown by analytical ultracentrifugation, there was no effect of guanidine/HCl concentrations up to 0.25 M on the quaternary structure of the enzyme in its oxidized and reduced forms: both sedimentation coefficient (S20,w = 4.9 +/- 0.1 S) and sedimentation equilibrium (75 +/- 3 kDa) yield the dimer. In the oxidized state the enzyme undergoes guanidine-dependent dissociation to the monomer with a midpoint of transition at 0.5 M. The kinetics of unfolding were found to be significantly faster for the reduced than for the oxidized enzyme. Renaturation and reactivation of reduced enzyme was more rapid and occurred with higher yields (100%) than for the oxidized enzyme (60-80% yield). Furthermore, the effect of denaturants on catalytic activity, and reductive activation of the oxidized form, were studied. Both increase in protein fluorescence and a stimulatory effect on the activities at low guanidine/HCl concentrations were observed for the oxidized and the reduced form of the enzyme. Denaturants increase the rate of reductive activation of NADP-malate dehydrogenase.     

Plasma protein carbonylation and physical exercise

Regular physical activity is associated with a reduced risk of coronary heart disease, as it probably modifies the balance between free-radical generation and antioxidant activity. On the other hand, however, acute physical activity increases oxygen uptake and leads to a temporary imbalance between the production of reactive oxygen and nitrogen species (RONS) and their disposal: this phenomenon is called oxidative stress. Proteins are one of the most important oxidation targets during physical exercise and carbonylation is one of the most common oxidative protein modifications. In cells there is a physiological level of oxidized proteins that doesn't interfere with cell function; however, an increase in oxidized protein levels may cause a series of cellular malfunctions that could lead to a disease state. For this reason the quantification of protein oxidation is important to distinguish a healthy state from a disease state. Several studies have demonstrated an increase of carbonylated plasma proteins in athletes after exercise, but none have identified targets of this oxidation. Recently a process of protein decarbonylation has been discovered, this may indicate that carbonylation could be involved in signal transduction. The aim of our research was to characterize plasma protein carbonylation in response to physical exercise in trained male endurance athletes. We analyzed by proteomic approach their plasma proteins at resting condition and after two different kinds of physical exercise (PE). We used 2D-GE followed by western blot with specific antibodies against carbonylated proteins. The 2D analysis identified Haptoglobin as potential protein target of carbonylation after PE. We also identified Serotransferrin and Fibrinogen whose carbonylation is reduced after exercise. These methods have allowed us to obtain an overview of plasma protein oxidation after physical exercise.     

Destabilization and denaturation of cellular protein by glutathione depletion

This investigation tested the hypothesis that depletion of intracellular glutathione, in contrast to its oxidation, could lead to non-native oxidation of protein thiols, thereby trapping proteins in an unstable conformation. Chinese hamster cells were exposed to the α,β-unsaturated dicarboxylic acid diethylmaleate in order to produce rapid gluthathione (GSH) depletion without oxidation. Measurement of the fluorescence of oxidized 2′,7′-dichlorofluorescein diacetate indicated that reactive oxygen species accumulated in GSH depleted cells. Glutathione depletion was found to alter protein thiol/disulfide exchange ratios such that 17 to 23 nmol of protein SH/mg protein underwent oxidation. Differential scanning calorimetry (DSC) of glutathione depleted cells yielded a profile of specific heat capacity versus temperature that was characteristic of cells containing destabilized and denatured protein. In addition, cells depleted of glutathione exhibited a two-fold increase in NP-40 insoluble protein. These results indicate that depletion of intracellular glutathione caused oxidation of protein thiols, protein denaturation and aggregation and provide a mechanism to explain how GSH depletion can initiate stress responses.