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1.
The endogenous neurotoxin, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), has been considered a potential causative factor for the pathogenesis of Parkinsonos disease (PD). In the present study, we examined the pattern of human Cu,Zn-superoxide dismutase (SOD) modification elicited by salsolinol. When Cu,Zn-SOD was incubated with salsolinol, some protein fragmentation and some higher molecular weight aggregates were occurred. Salsolinol led to inactivation of Cu,Zn-SOD in a concentration-dependent manner. Free radical scavengers and catalase inhibited the salsolinolmediated Cu,Zn-SOD modificaiton. Exposure of Cu,Zn-SOD to salsolinol led also to the generation of protein carbonyl compounds. The deoxyribose assay showed that hydroxyl radicals were generated during the oxidation of salsolinol in the presence of Cu,Zn-SOD. Therefore, the results indicate that free radical may play a role in the modification and inactivation of Cu,Zn-SOD by salsolinol.  相似文献   

2.
Kim NH  Jeong MS  Choi SY  Hoon Kang J 《Biochimie》2004,86(8):553-559
Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for their survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of Cu,Zn-superoxide dismutase (SOD) in the modification of NF-L. When disassembled NF-L was incubated with Cu,Zn-SOD and H2O2, the aggregation of protein was proportional to the concentration of hydrogen peroxide. Cu,Zn-SOD/H2O2-mediated modification of NF-L was significantly inhibited by radical scavenger, spin trap agents and copper chelators. Dityrosine crosslink formation was obtained in Cu,Zn-SOD/H2O2-mediated NF-L aggregates. Antioxidant molecules, carnosine and anserine significantly inhibited the aggregation of NF-L and the formation of dityrosine. This study suggests that copper-mediated NF-L modification may be closely related to oxidative reactions which play a critical role in neurodegenerative diseases.  相似文献   

3.
The peroxidase activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) has been extensively studied in recent years due to its potential relationship to familial amyotrophic lateral sclerosis. The mechanism by which Cu,Zn-SOD/hydrogen peroxide/bicarbonate is able to oxidize substrates has been proposed to be dependent on an oxidant whose nature, diffusible carbonate radical anion or enzyme-bound peroxycarbonate, remains debatable. One possibility to distinguish these species is to examine whether protein targets are oxidized to protein radicals. Here, we used EPR methodologies to study bovine serum albumin (BSA) oxidation by Cu,Zn-SOD/hydrogen peroxide in the absence and presence of bicarbonate or nitrite. The results showed that BSA oxidation in the presence of bicarbonate or nitrite at pH 7.4 produced mainly solvent-exposed and -unexposed BSA-tyrosyl radicals, respectively. Production of the latter was shown to be preceded by BSA-cysteinyl radical formation. The results also showed that hydrogen peroxide/bicarbonate extensively oxidized BSA-cysteine to the corresponding sulfenic acid even in the absence of Cu,Zn-SOD. Thus, our studies support the idea that peroxycarbonate acts as a two-electron oxidant and may be an important biological mediator. Overall, the results prove the diffusible and radical nature of the oxidants produced during the peroxidase activity of Cu,Zn-SOD in the presence of bicarbonate or nitrite.  相似文献   

4.
Alpha-synuclein is a major component of the abnormal protein aggregation in Lewy bodies of Parkinson's disease (PD) and senile plaques of Alzheimer's disease (AD). Previous studies have shown that the aggregation of alpha-synuclein was induced by copper (II) and H(2)O(2) system. Since copper ions could be released from oxidatively damaged Cu,Zn-superoxide dismutase (SOD), we investigated the role of Cu,Zn-SOD in the aggregation of alpha-synuclein. When alpha-synuclein was incubated with both Cu,Zn-SOD and H(2)O(2), alpha-synuclein was induced to be aggregated. This process was inhibited by radical scavengers and spin trapping agents such as 5,5'-dimethyl 1-pyrolline N-oxide and tert-butyl-alpha-phenylnitrone. Copper chelators, diethyldithiocarbamate and penicillamine, also inhibited the Cu,Zn-SOD/H(2)O(2) system-induced alpha-synuclein aggregation. These results suggest that the aggregation of alpha-synuclein is mediated by the Cu,Zn-SOD/H(2)O(2) system via the generation of hydroxyl radical by the free radical-generating function of the enzyme. The Cu,Zn-SOD/H(2)O(2)-induced alpha-synuclein aggregates displayed strong thioflavin-S reactivity, reminiscent of amyloid. These results suggest that the Cu,Zn-SOD/H(2)O(2) system might be related to abnormal aggregation of alpha-synuclein, which may be involved in the pathogenesis of PD and related disorders.  相似文献   

5.
Jung Hoon Kang 《BMB reports》2013,46(11):555-560
Acrolein is the most reactive aldehydic product of lipid peroxidation and is found to be elevated in the brain when oxidative stress is high. The effects of acrolein on the structure and function of human Cu,Zn-superoxide dismutase (SOD) were examined. When Cu,Zn-SOD was incubated with acrolein, the covalent crosslinking of the protein was increased, and the loss of enzymatic activity was increased in a dose-dependent manner. Reactive oxygen species (ROS) scavengers and copper chelators inhibited the acrolein-mediated Cu,Zn-SOD modification and the formation of carbonyl compound. The present study shows that ROS may play a critical role in acrolein-induced Cu,Zn-SOD modification and inactivation. When Cu,Zn-SOD that has been exposed to acrolein was subsequently analyzed by amino acid analysis, serine, histidine, arginine, threonine and lysine residues were particularly sensitive. It is suggested that the modification and inactivation of Cu,Zn-SOD by acrolein could be produced by more oxidative cell environments. [BMB Reports 2013; 46(11): 555-560]  相似文献   

6.
Carnosine (beta-alanyl-L-histidine), homocarnosine (gamma-amino-butyryl-L-histidine) and anserine (beta-alanyl-1-methyl-L-histidine) have been proposed to act as anti-oxidants in vivo. The protective effects of carnosine and related compounds against the oxidative damage of human Cu,Zn-superoxide dismutase (SOD) by peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) were studied. The oxidative damage to Cu,Zn-SOD by AAPH-derived radicals led to protein fragmentation, which is associated with the inactivation of enzyme. Carnosine, homocarnosine and anserine significantly inhibited the fragmentation and inactivation of Cu,Zn-SOD by AAPH. All three compounds also inhibited the release of copper ions from the enzyme and the formation of carbonyl compounds in AAPH-treated Cu,Zn-SOD. These compounds inhibited the fragmentation of other protein without copper ion. The results suggest that carnosine and related compounds act as the copper chelator and peroxyl radical scavenger to protect the protein fragmentation. Oxidation of amino acid residues in Cu,Zn-SOD induced by AAPH were significantly inhibited by carnosine and related compounds. It is proposed that carnosine and related dipeptides might be explored as potential therapeutic agents for pathologies that involve Cu,Zn-SOD modification mediated by peroxyl radicals.  相似文献   

7.
Methylglyoxal (MG) has been identified as an intermediate in non-enzymatic glycation, and increased levels have been reported in patients with diabetes. In this study, the effect of MG on the structure and function of human Cu,Zn-superoxide dismutase (SOD) was investigated. MG modifies Cu,Zn-SOD, as indicated by the formation of fluorescent products. When Cu, Zn-SOD was incubated with MG, covalent crosslinking of the protein increased progressively. MG-mediated modification of Cu,Zn-SOD led to loss of enzymatic activity and release of copper ions from the protein. Radical scavengers inhibited the crosslinking of Cu,Zn-SOD. When Cu,Zn-SOD that had been exposed to MG was analyzed, glycine, histidine, lysine, and valine residues were found to be particularly sensitive. It is suggested that oxidative damage to Cu,Zn-SOD by MG may perturb cellular antioxidant defense systems and damage cells. This effect may account, in part, for organ deterioration in diabetes.  相似文献   

8.
Cu,Zn-superoxide dismutase (SOD) can catalyze hydroxyl radical generation using H2O2 as a substrate. Lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system was investigated. When linoleic acids micelles or phosphatidylcholine liposomes were incubated with Cu,Zn-SOD and H2O2, lipid peroxidation was gradually increased in a time-dependent manner. The extent of lipid peroxidation was proportional to Cu,Zn-SOD and H2O2 concentrations. Hydroxyl radical scavengers and copper chelator inhibited lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system. These results suggest that lipid peroxidation is mediated by the Cu,Zn-SOD and H2O2 system via the generation of hydroxyl radicals by a combination of the peroxidative reaction of Cu,Zn-SOD and the Fenton-like reaction of free copper released from oxidatively damaged SOD.  相似文献   

9.
Cu,Zn-Superoxide dismutase (SOD) was isolated from the liver of 3-, 12-, and 26-month-old Fisher 344 (F344) rats. Specific activity and metal content of the enzyme, purified by ion-exchange and size-exclusion chromatography, did not significantly change with age. Electrospray ionization-mass spectrometry and amino acid analysis of Cu,Zn-SOD apoprotein, further purified by reverse-phase HPLC, showed neither significant loss of amino acids nor accumulation of oxidized isoforms with age. When bovine Cu,Zn-SOD, oxidized with H(2)O(2) in vitro, was added to rat liver homogenate, we reisolated circa 70% of the oxidized bovine Cu,Zn-SOD together with the rat isoform, showing that oxidized Cu,Zn-SOD can be recovered from tissue homogenate. Therefore, our data do not confirm an earlier hypothesis that oxidatively modified Cu,Zn-SOD protein accumulates in the liver of aged F344 rats.  相似文献   

10.
Superoxide radicals are known to inhibit progesterone production by luteal cells and have also been reported to cause apoptosis in various cells. The corpus luteum has an antioxidant enzyme to scavenge superoxide radicals: copper-zinc superoxide dismutase (Cu, Zn-SOD). However, it remains unknown how the decrease in intracellular Cu,Zn-SOD activity influences luteal function. This study was therefore undertaken to investigate whether suppression of intracellular Cu,Zn-SOD activity inhibits progesterone production by rat luteal cells and causes apoptosis. To suppress intracellular Cu, Zn-SOD activity, dispersed rat luteal cells were incubated with Cu, Zn-SOD antisense oligonucleotides. The 48-h treatment with antisense oligonucleotides (10 microM) inhibited Cu,Zn-SOD activity by 50% and Cu,Zn-SOD mRNA level by 30%, whereas sense oligonucleotides used as the control had no effect. Progesterone concentration in the medium was significantly decreased by the 48-h treatment with antisense oligonucleotides in the presence of hCG, and this inhibitory effect was completely blocked by the simultaneous addition of N-acetyl-L-cysteine, an antioxidant. Treatment with antisense oligonucleotides caused no significant change in the percentage of apoptotic cells as morphologically evaluated by the nuclear staining with Hoechst dye. In conclusion, the decrease in intracellular Cu, Zn-SOD activities inhibits progesterone production by rat luteal cells, which may be mediated by superoxide radicals, suggesting that intracellular Cu,Zn-SOD plays important roles in the regulation of luteal function.  相似文献   

11.
The effects of administration of oxidized rapeseed oil and α-lipoic acid on activities of blood antioxidant enzymes and malondialdehyde (MDA) concentration were studied in laboratory rats fed a high-fat diet. Addition of oxidized oil resulted in increased production of oxygen radicals, evidenced by elevated plasma MDA production. Such effect was counteracted by administration of α-lipoic acid. There was an increase of the activities of superoxide dismutase (total and Cu/Zn-SOD) and catalase in rats fed a high-fat diet to which 10% oxidized oil was added. Administration of α-lipoic acid resulted in a decrease of the activities of these enzymes.  相似文献   

12.
Kang JH 《BMB reports》2012,45(2):114-119
Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a compound derived from dopamine metabolism and is capable of causing dopaminergic neurodegeneration. Oxidative modification of neurofilament proteins has been implicated in the pathogenesis of neurodegenerative disorders. In this study, oxidative modification of neurofilament-L (NF-L) by salsolinol and the inhibitory effects of histidyl dipeptides on NF-L modification were investigated. When NF-L was incubated with 0.5 mM salsolinol, the aggregation of protein was increased in a time-dependent manner. We also found that the generation of hydroxyl radicals (?OH) was linear with respect to the concentrations of salsolinol as a function of incubation time. NF-L exposure to salsolinol produced losses of glutamate, lysine and proline residues. These results suggest that the aggregation of NF-L by salsolinol may be due to oxidative damage resulting from free radicals. Carnosine, histidyl dipeptide, is involved in many cellular defense processes, including free radical detoxification. Carnosine, and anserine were shown to significantly prevent salsolinol- mediated NF-L aggregation. Both compounds also inhibited the generation of ?OH induced by salsolinol. The results indicated that carnosine and related compounds may prevent salsolinol-mediated NF-L modification via free radical scavenging.  相似文献   

13.
Superoxide dismutase in vesicular arbuscular-mycorrhizal red clover plants   总被引:5,自引:0,他引:5  
The isoenzymatic pattern of Superoxide dismutase (SOD; EC 1.15.1.1) was studied in the symbiosis of Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe-Trifolium prarense L A Cu.Zn-SOD (M, 40500) was found in spores of G. mosseae . while one Mn-SOD (Mn-SOD I) and two Cu.Zn-SODs (Cu.Zn-SOD 1 and Cu.Zn-SOD II) were present in both roots and leaves of T. pratense . Molecular masses for Cu.Zn-SOD I and Cu.Zn-SOD II were 31000 and 34300. respectively. However, when T. prateme and G. mosseae were associated, mycorrhizal roots showed two new iso-zymes, Mn-SOD II and mycCu.Zn-SOD, which have relative molecular masses of 37 800 and 33 300, respectively. The mycCu.Zn-SOD was found to be specific for this association, whereas Mn-SOD II was also present in nodules of Rhizobium-T. pra-tense . Results suggest that both enzymes are induced in the T. praiense roots in response to invasion by mycorrhizal fungi, perhaps as a result of an increase in the generation of O-2 radicals in plant roots.  相似文献   

14.
The senescence-accelerated prone mouse strain 8 (SAMP8) exhibits a remarkable age-accelerated deterioration in learning and memory. In this study, we identified carbonyl modification, a marker of protein oxidation, in liver and brain of SAMP8 from peptide mass fingerprints using MALDI-TOF mass spectrometry in combination with LC-MS/MS analysis. Carbonyl modification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in liver at 3 month and hippocampal cholinergic neurostimulating peptide precursor protein (HCNP-pp) in brain at 9 month were higher in SAMP8 compared with control SAMR1. We demonstrated carbonyl modification of purified Cu,Zn-SOD increased by the reaction with H2O2. Therefore, progressive accumulation of oxidative damage to Cu,Zn-SOD, may cause dysfunction of defense systems against oxidative stress in SAMP8 with a higher oxidative states, leading to acceleration of aging. Furthermore, carbonyl modification of HCNP-pp may be involved in pathophysiological alterations associated with deterioration in the learning and memory in the brain seen in SAMP8.  相似文献   

15.
Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide. In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was active in vitro and was more resistant to inactivation by tert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.  相似文献   

16.
Copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese superoxide dismutase (Mn-SOD) in some model experiments in vitro demonstrated antioxidant as well as pro-oxidant properties. In the present study, yeast Saccharomyces cerevisiae lacking Mn-SOD were studied using Cu,Zn-SOD inhibitor N-N'-diethyldithiocarbamate (DDC) as a model system to study the physiological role of the yeast Cu,Zn-SOD. Yeast treatment by DDC caused dose-dependent inhibition of SOD in vivo, with 75% inhibition at 10mM DDC. The inhibition of SOD by DDC resulted in modification of carbonylprotein levels, indicated by a bell-shaped curve. The activity of glutathione reductase, isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase (enzymes associated with antioxidant) increased, demonstrating a compensatory effect in response to SOD inhibition by different concentrations of DDC. A strong positive correlation (R2=0.97) was found between SOD and catalase activities that may be explained by the protective role of SOD for catalase. All observed effects were absent in the isogenic SOD-deficient strain that excluded direct DDC influence. The results are discussed from the point of view that in vivo Cu,Zn-SOD of S. cerevisiae can demonstrate both anti- and pro-oxidant properties.  相似文献   

17.
Abstract: Copper/zinc superoxide dismutase (Cu/Zn-SOD) is a major free radical scavenging enzyme. Increased Cu/Zn-SOD activity protects cells against oxidative stress mediated by different mechanisms. However, there is also in vitro and in vivo evidence that, in the absence of abnormal oxidative stress, chronic increased Cu/Zn-SOD activity is detrimental to living cells. To address this issue, we examined the fate of mature midbrain neurons from transgenic mice expressing human Cu/Zn-SOD and from their nontransgenic littermates. Midbrain from transgenic pups had about threefold higher Cu/Zn-SOD activity than that from nontransgenic pups. Virtually all transgenic neurons were strongly immunoreactive for human Cu/Zn-SOD protein in their cell bodies and processes. The number of midbrain neurons decreased over time in both transgenic and nontransgenic cultures, but to a significantly smaller extent in the transgenic cultures. Postnatal midbrain neurons died by either necrosis or apoptosis, and increased Cu/Zn-SOD activity attenuated both forms of cell death. Furthermore, increased Cu/Zn-SOD activity better prevented the loss of dopaminergic neurons than GABAergic neurons. We also found that neuronal processes were dramatically denser in transgenic cultures than in nontransgenic cultures. These results indicate that chronic increased Cu/Zn-SOD activity does not appear to be detrimental, but rather promotes cell survival and neuronal process development in postnatal midbrain neurons, probably by providing more efficient detoxification of free radicals. They also show that increased Cu/Zn-SOD activity does not seem to play a critical role in determining the mode of cell death in this culture system.  相似文献   

18.
9,10-Phenanthraquinone (PQ), a major quinone contained in diesel exhaust particles and atmospheric PM(2.5), undergoes one-electron reduction by flavin enzymes such as NADPH-cytochrome P450 reductase, leading to production of reactive oxygen species in vitro. We have detected an ESR signal for superoxide (O(2)(-)) and hydroxyl radicals ((.)OH) by the spin trap method when PQ was mixed with P450 reductase, NADPH, and iron(III). When we examined the effects of PQ on A549 human pulmonary epithelial cells, PQ induced apoptosis with a LC(50) of approximately 7 microM. Formation of protein carbonyls was also detected in cells after treatment with PQ, suggesting that PQ induces oxidative damage. Iron chelators such as 1,10-phenanthroline (OP), desferrioxamine mesylate, and deferiprone respectively afforded protection against the toxic effects of PQ. Furthermore, treatment of A549 cells with 10-20 microM PQ for 12 h specifically down-regulated protein levels of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and heme oxygenase-1 (HO-1) by more than 50%. Pretreatment of cells with OP (10 microM) markedly reduced the down-regulation of Cu,Zn-SOD and HO-1 and protein carbonyl formation in response to PQ. The inhibitor of Cu,Zn-SOD, diethyldithiocarbamate, enhanced the toxic effects of 5 microM PQ. The present findings suggest that PQ causes iron-mediated oxidative damage that is exacerbated by the concomitant down-regulation of Cu,Zn-SOD.  相似文献   

19.
Abstract: The Gly93→Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (~50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100–120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.  相似文献   

20.
To investigate the role of superoxide dismutase (SOD) in the ovulatory process, SOD isozymes and their mRNAs were determined in the ovary of 22-day-old rats. After treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), ovarian activity of Mn-SOD decreased markedly while Cu/Zn-SOD remained unchanged. However, the ovarian level of mRNA for Mn-SOD markedly increased after hCG-treatment while that for Cu/Zn-SOD decreased only slightly. Ovulation was inhibited by intravenous injection of a long-acting SOD. These results suggested that superoxide radicals in the ovary might play a critical role in the mechanism for hCG-induced ovulation.  相似文献   

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