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1.
Although more than 100 mutations have been identified in the copper/zinc superoxide dismutase (Cu/Zn-SOD) in familial amyotrophic lateral sclerosis (FALS), the mechanism responsible for FALS remains unclear. The finding of the present study shows that FALS-causing mutant Cu/Zn-SOD proteins (FALS mutant SODs), but not wild-type SOD, are barely detected by three monoclonal antibodies (mAbs) in Western blot analyses. The enzyme-linked immunosorbent assay for denatured FALS mutant SODs by dithiothreitol, SDS, or heat treatment also showed a lowered immunoreactivity against the mAbs compared with wild-type SOD. Because all the epitopes of these mAbs are mapped within the Greek key loop (residues 102-115 in human Cu/Zn-SOD), these data suggest that different conformational changes occur in the loop between wild-type and FALS mutant SODs during the unfolding process. Circular dichroism measurements revealed that the FALS mutant SODs are sensitive to denaturation by dithiothreitol, SDS, or heat treatment, but these results do not completely explain the different recognition by the mAbs between wild-type and FALS mutant SODs under the denatured conditions. The study on the conformational changes in local areas monitoring with mAbs may provide a new insight into the etiology of FALS.  相似文献   

2.
The kinetics of bovine Cu,Zn superoxide dismutase were studied by pulse radiolysis. To ensure the absence of catalytically active free copper, commercially obtained holo-superoxide dismutase was demetallated, and the apo-superoxide dismutase concentrations were determined by isothermal titration calorimetry prior to reconstitution with defined amounts of copper and zinc. The catalytic rate constant was determined as a function of ionic strength over the range of 4-154 mM, and of the copper and zinc content. The catalytic rate constant increases with ionic strength up to (1.5 +/- 0.2) x 10(9) M(-1) s(-1) at an ionic strength of 15 mM, and then decreases. At pH 7 and 50 mM ionic strength, k = (1.2 +/- 0.2) x 10(9) M(-1) s(-1), and at a physiologically relevant ionic strength of 150 mM, it is (0.7 +/- 0.1) x 10 (9) M(-1) s(-1). The effect of ionic strength is ascribed to the inhomogeneous electric field generated by the surface charges of superoxide dismutase. The value of the catalytic rate constant at 50 mM is ca. 2-fold smaller than earlier values reported in the literature. The relationship between copper content and the catalytic rate constant shows that addition of more than a stoichiometric amount of copper cannot be masked efficiently by EDTA. The possibility exists that earlier reported values were based on experiments contaminated with trace amounts of copper.  相似文献   

3.
We report herein that the level of reactive oxygen species (ROS) observed using dihydrorhodamine is much higher in either GTS1-deleted (gts1Delta) or GTS1-overexpressing (TMpGTS1) transformants than in the wild-type and that the levels of protein carbonyls are increased and the glutathione levels are decreased in both transformants. Consistently, the activities of superoxide dismutases (SODs) in both gts1Delta and TMpGTS1 were severely weakened, while the protein levels of both Cu/Zn-SOD and Mn-SOD were not so changed. As the intracellular copper levels were significantly increased in both transformants, we hypothesized that, in either gts1Delta or TMpGTS1 cells, the imbalanced homeostasis of copper induced an accumulation of ROS which caused inactivation of SODs further increasing ROS levels.  相似文献   

4.
Superoxide dismutases (SODs; EC 1.15.1.1) play important roles in the protection of the parasites against cellular oxygen-mediated killing of the hosts. A copper/zinc-containing SOD (Cu/Zn-SOD) was identified previously from lung fluke, Paragonimus westermani. To expand our understanding of P. westermani SOD, we isolated a complementary DNA encoding a Cu/Zn-SOD, expressed the active enzyme in Escherichia coli, and characterized its biochemical properties. The deduced amino acid (aa) sequence of the gene shared up to 73.7% identities with Cu/Zn-SODs of other helminths and shared well-conserved characteristic motifs and essential aa residues involved in coordinating copper and zinc enzymatic functions. Recombinant Cu/ Zn-SOD exhibited comparable biochemical properties with that of the native enzyme, including pH optima and potassium cyanide-and hydrogen peroxide-sensitive inhibition profiles. The active enzyme consisted of 2 identical subunits covalently linked by disulfide bonds. The enzyme was constitutively expressed throughout various developmental stages of the parasite. The levels increased as P. westermani matured and plateaued in adult stage. Our result suggests the enzyme might play an important role for parasites to survive in the hosts through its superoxide anion-detoxifying function.  相似文献   

5.
The relationships among concentrations of copper and zinc, the oxidase activity of ceruloplasmin (Cp) in serum, and Cu,Zn-SOD (superoxide dismutase) activity in erythrocytes were investigated in men with atherosclerosis obliterans (AO) and a control group. The oxidase activity of Cp was measured with o-dianisidine dihydrochloride as a substrate, and Cu,Zn-SOD activity in erythrocytes by using the RANSOD kit. The lipid profile and uric acid concentration were determined in AO and control groups. The results showed higher copper and zinc concentrations in serum in the AO group (20.0±3.5 and 18.0±3.2 μmol/L, respectively) in comparison with the control group (15.6±2.3 and 14.7±1.9 μmol/L). The Cp activity in serum was higher in the AO group (174.2±61.8 U/L) than in the control group (93.7±33.9 U/L), and a significant difference was found in the activity of Cu,Zn-SOD in erythrocytes (2389±1396 and 1245±365 U/g Hb, respectively) between both groups. The activity of Cu,Zn-SOD was positively correlated with copper in the control group (r=0.73), but not in AO, and negatively with uric acid concentration (r=−0.63) in the AO group. The oxidase activity of Cp was correlated with copper, but not zinc, in AO and control groups (r≥0.65). Negative correlation coefficients were calculated for uric acid and copper and zinc concentrations in the AO group (−r≥0.61). Increased copper concentrations and oxidase activity of Cp in serum in AO and the activity of Cu,Zn-SOD in erythrocytes could result from atherosclerotic disease, accompanied by chronic ischemia of a lower limb. These results suggest also that relationship between copper concentration and Cu,Zn-SOD activity in erythrocytes found in the serum of healthy subjects may be disturbed in pathologic conditions.  相似文献   

6.
7.
Nitric oxide (*NO) and its by-products modulate many physiological functions of skeletal muscle including blood flow, metabolism, glucose uptake, and contractile function. However, growing evidence suggests that an overproduction of nitric oxide contributes to muscle wasting in a number of pathologies including chronic heart failure, sepsis, COPD, muscular dystrophy, and extreme disuse. Limited data point to the potential of inhibition various enzymes by reactive nitrogen species (RNS), including (.)NO and its downstream products such as peroxynitrite, primarily in purified systems. We hypothesized that exposure of skeletal muscle to RNS donors would reduce or downregulate activities of the crucial antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). Diaphragm muscle fiber bundles were extracted from 4-month-old Fischer-344 rats and, in a series of experiments, exposed to either (a) 0 (control), 1, or 5 mM diethylamine NONOate (DEANO: *NO donor); (b) 0, 100, 500 microM, or 1 mM sodium nitroprusside (SNP: *NO donor); (c) 0 or 2 mM S-nitroso-acetylpenicillamine (SNAP: *NO donor); or (d) 0 or 500 microM SIN-1 (peroxynitrite donor) for 60 min. DEANO resulted in a 50% reduction in CAT, GPX, and a dose-dependent inhibition of Cu, Zn-SOD. SNP resulted in significantly lower activities for total SOD, Mn-SOD isoform, Cu, Zn-SOD isoform, CAT, and GPX in a dose-dependent fashion. Two millimolar SNAP and 500 microM SIN-1 also resulted in a large and significant inhibition of total SOD and CAT. These data indicate that reactive nitrogen species impair antioxidant enzyme function in an RNS donor-specific and dose-dependent manner and are consistent with the hypothesis that excess RNS production contributes to skeletal muscle oxidative stress and muscle dysfunction.  相似文献   

8.
The role of the Lys68*:Glu265 intersubunit salt bridge that is conserved (Csb) in all known aspartate aminotransferases (AATases), except those of animal cytosolic, Ac (His68*:Glu265), and plant mitochondrial, Pm (Met68*:Gln265), origins, was evaluated in the Escherichia coli AATase. Two double-mutant cycles, to K68M/E265Q and the charge reversed K68E/E265K, were characterized with the context dependence (C) and impact (I) formalism, previously defined for functional chimeric analysis. Mutations of Lys68* with Glu265 fixed are generally more deleterious than the converse mutations of Glu265 with Lys68* fixed, showing that buried negative charges have greater effects than buried positive charges in this context. Replacement of the charged Lys68*:Glu265 with the K68M/E265Q neutral pair introduces relatively small effects on the kinetic parameters. The differential sensitivity of k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) to salt bridge mutagenic replacements is shown by a linear-free energy relationship, in which the logarithms of the latter second order rate constants are generally decreased by a factor of two more than are those of the former. Thus, k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) are 133 and 442 mM(-1)s(-1) for the wild-type (WT) enzyme, respectively, but their relative order is reversed in the more severely compromised mutants (14.8 and 5.3 mM(-1)s(-1) for K68E). A Venn diagram illustrates apparent forced covariances of groups of amino acids that accompany the naturally occurring salt bridge replacements in the Pm and Ac classes. The more deeply rooted tree indicates that the Csb variant was the ancestral specie.  相似文献   

9.
We discovered that a mutant strain of the dimorphic yeast Yarrowia lipolytica could grow in the yeast form in high concentrations of copper sulfate. The amount of metal accumulated by Y. lipolytica increased with increasing copper concentrations in the medium. Washing with 100 mM EDTA released at least 60% of the total metal from the cells, but about 20–25 μmol/g DW persisted, which represented about 30% of the soluble fraction of cultured cells. The soluble fraction (mainly cytosol) contained only about 10% of the total metal content within cells cultured in medium supplemented with 6 mM copper. We suggest that although a high copper concentration induces an efflux mechanism, the released copper becomes entrapped in the periplasm and in other parts of the cell wall. Washing with EDTA liberated not only copper ions, but also melanin, a brown pigment that can bind metal and which located at the cell wall. These findings indicated that melanin participates in the mechanism of metal accumulation. Culture in medium supplemented with copper obviously enhanced the activities of Cu, Zn-SOD, but not of Mn-SOD.  相似文献   

10.
Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is a ubiquitous enzyme with an essential role in antioxidant defense. To better understand structural factors at the origin of the highly efficient superoxide dismutation mechanism, we analyzed the consequence of copper reduction on the electronic properties of the backbone and individual amino acids by using electrochemistry coupled to Fourier transform infrared spectroscopy. Comparison of data recorded with bovine erythrocyte and recombinant chloroplastic Cu,Zn-SOD from Lycopersicon esculentum, expressed as a functional tetramer in Escherichia coli and (14)N- or fully (15)N-labeled, demonstrated that the infrared changes were dominated by reorganizations of peptide bonds and histidine copper ligands. Two main infrared modes of histidine side chain, markers of metal coordination, were identified by using Cu- and Zn-methylimidazole models: the nu(C(4)C(5))at 1605-1594 cm(-1) or approximately 1586 cm(-1) for Ntau or Npi coordination, and the nu(C(5)Ntau) at approximately 1113-1088 cm(-1). These modes, also identified in Cu,Zn-SOD by using (15)N labeling, showed that the electronic properties of the histidine Ntau ligands of copper are mostly affected upon copper reduction. A striking conclusion of this work is that peptide groups from loops and beta-sheet largely participate in charge redistribution upon copper reduction, and in contrast, electronic properties of polar and charged amino acids of the superoxide access channel remain unaffected. This is notably shown for the strictly conserved Arg-143 by site-directed mutagenesis on chloroplastic Cu,Zn-SOD. Charge compensation by the peptide backbone and preserved electronic properties of the superoxide access channel and docking site upon copper reduction may be the determinant factors for the high reaction kinetics of superoxide with both reduced and oxidized Cu,Zn-SOD.  相似文献   

11.
The isoenzyme I of cytosolic Cu,Zn-superoxide dismutase (SOD) from Nicotiana plumbaginifolia (tobacco) leaves has been purified to apparent homogeneity. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electrophoresis shows that the enzyme is composed of two equal subunits of 16.6 kDa The isolectric point, assayed by isoelectric focusing, in the pH range of 3.5-6.5, is 4.3. The enzyme stability was tested at different temperatures, pH, and concentration of inhibitors (KCN and H(2)O(2)). The catalytic constant (k(cat)) was 1.17 +/- 0.14 x 10(9) M(-1) s(-1) at pH 9.9 and 0.1 M ionic strength. The activation energy of the thermal denaturation process is 263 kJ mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn-SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino acid substitution around the active sites.  相似文献   

12.
A kinetic study of the oxidation of the copper(I) form of the blue copper protein stellacyanin (St(I) by Co(EDTA)-- has been performed. Observed rate constants approach a saturation limit with increasing [Co(EDTA)--] at pH 7, consistent with a mechanism involving rapid pre-equilibrium oxidant-protein complex formation followed by rate-limiting intramolecular Cu(I) to Co(III) electron transfer: Co(EDTA)-- + St(i Qp in equilibrium Co(EDTA)-- ---St(I) Co(EDTA)-- ---St(I) k2 leads to Co(EDTA)2-- ---St(II) (Qp = 149 M--1, k2 = 0.169 sec--1; 25.1 degrees, pH 7.0 mu 0.5 M (phosphate)). Activation parameters based on k2 (deltaH not equal to = 1.8 kcal/mol, deltaS not equal to = --56 cal/mol-deg) indicate that the electron transfer process is substantially nondiabatic, in marked contrast with results obtained for Co(phen) 3 3+ as the oxidant. Linear kobsd VS. [Co(EDTA)--] plots are reported for the Co(EDTA)-- oxidation of cuprous stellacyanin at pH 10 (k = 8.9 M--1 sec--1; 25.0, pH 10, mu 0.5 M (carbonate); DELTaH not equal to 11.3 kcal/mol, deltaS not equal to = -16 cal/mol-deg) and at pH 7 in the presence of excess EDTA (k = 21.2 M--1 sec--1; 25.1 degree, pH 7.0, mu 0.5 M (phosphate), [EDTA] tot = 5 X 10(--4) M; deltaH not equal to = 5.9 kcal/mol, delta S not equal to = --33 cal/mol-deg). It is concluded that Co(EDTA)-- adopts an electron transfer mechanism similar to that preferred by Co(phen)33+ under conditions where the oxidant is prevented from binding strongly to reduced stellacyanin.  相似文献   

13.
Nitroxyl (NO(-)) may be produced by nitric-oxide synthase and by the reduction of NO by reduced Cu,Zn-SOD. The ability of NO(-) to cause oxidations and of SOD to inhibit such oxidations was therefore explored. The decomposition of Angeli's salt (AS) produces NO(-) and that in turn caused the aerobic oxidation of NADPH, directly or indirectly. O(2) was produced concomitant with the aerobic oxidation of NADPH by AS, as evidenced by the SOD-inhibitable reduction of cytochrome c. Both Cu,Zn-SOD and Mn-SOD inhibited the aerobic oxidation of NADPH by AS, but the amounts required were approximately 100-fold greater than those needed to inhibit the reduction of cytochrome c. This inhibition was not due to a nonspecific protein effect or to an effect of those large amounts of the SODs on the rate of decomposition of AS. NO(-) caused the reduction of the Cu(II) of Cu,Zn-SOD, and in the presence of O(2), SOD could catalyze the oxidation of NO(-) to NO. The reverse reaction, i.e. the reduction of NO to NO(-) by Cu(I),Zn-SOD, followed by the reaction of NO(-) with O(2) would yield ONOO(-) and that could explain the oxidation of dichlorofluorescin (DCF) by Cu(I),Zn-SOD plus NO. Cu,Zn-SOD plus H(2)O(2) caused the HCO(3)(-)-dependent oxidation of DCF, casting doubt on the validity of using DCF oxidation as a reliable measure of intracellular H(2)O(2) production.  相似文献   

14.
Superoxide dismutases: active sites that save, but a protein that kills   总被引:4,自引:0,他引:4  
Protection from oxidative damage is sufficiently important that biology has evolved three independent enzymes for hastening superoxide dismutation: the Cu- and Zn-containing superoxide dismutases (Cu,Zn-SODs), the SODs that are specific for Fe or Mn or function with either of the two (Fe-SODs, Mn-SODs or Fe/Mn-SODs), and the SODs that use Ni (Ni-SODs). Despite the overwhelming similarities between the active sites of Fe-SOD and Mn-SOD, the mechanisms and redox tuning of these two sites appear to incorporate crucial differences consistent with the differences between Fe3+/2+ and Mn3+/2+. Ni-SOD is revealed by spectroscopy to employ completely different ligation to that of the other SODs while nonetheless incorporating a device also found in Cu,Zn-SOD. Finally, the protein of human Cu,Zn-SOD appears to be an important contributor to the development of amyotrophic lateral sclerosis, possibly because of its propensity for extended beta-sheet formation.  相似文献   

15.
The fragmentation of human Cu,Zn-superoxide dismutase (SOD) was observed during incubation with H(2)O(2). Hydroxyl radical scavengers such as sodium azide, formate and mannitol protected the fragmentation of Cu,Zn-SOD. These results suggested that *OH was implicated in the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation. Carnosine, homocarnosine and anserine have been proposed to act as anti-oxidants in vivo. We investigated whether three compounds could protect the fragmentation of Cu,Zn-SOD induced by H(2)O(2). The results showed that carnosine, homocarnosine and anserine significantly protected the fragmentation of Cu,Zn-SOD. All three compounds also protected the loss of enzyme activity induced by H(2)O(2). Carnosine, homocarnosine and anserine effectively inhibited the formation of *OH by the Cu,Zn-SOD/H(2)O(2) system. These results suggest that carnosine and related compounds can protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation through the scavenging of *OH.  相似文献   

16.
Rat liver was homogenized in isotonic buffer, fractionated by differential centrifugation, and then subfractionated by equilibrium sedimentation in Nycodenz gradients. Fractions were assayed for both Cu,Zn-superoxide dismutase (SOD) and Mn-SOD by exploiting the cyanide sensitivity of the former activity and by the use of specific antibodies. As expected, the cytosol and lysosomal fractions contained Cu,Zn-SOD; while the mitochondrial matrix contained Mn-SOD. In mitochondria, Cu,Zn-SOD was found in the intermembrane space and Mn-SOD in the matrix and also on the inner membrane. The Mn-SOD associated with the inner membrane was solubilized by 0.5 m NaCl. Surprisingly the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be solubilized with a detergent, and to lesser degree with 0.5 m NaCl. Both the cytosolic and mitochondrial Cu,Zn-SODs were isolated and compared. They have identical molecular mass, cyanide sensitivity, SDS sensitivity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn-SOD knockout mice was entirely devoid of Cu,Zn-SOD; indicating that the cytosolic and the intermembrane space Cu,Zn-SODs are coded for by the same gene. The significance of this distribution of the SODs is discussed.  相似文献   

17.
Eight mutant Cu,Zn-superoxide dismutases (SODs) related to familial amyotrophic lateral sclerosis (FALS) were produced in a baculovirus/insect cell expression system and their molecular properties in terms of hydroxyl radical formation and aggregation were compared with the wild-type enzyme. Treatment of the enzymes with Chelex 100 resin decreased Cu contents as well as SOD activities in all mutant Cu,Zn-SODs, indicating that the affinities of the enzymes for copper ion were decreased. Contrary to previous reports, all the mutant Cu,Zn-SODs exhibited less reactive oxidant producing ability in the presence of hydrogen peroxide than the wild-type enzyme. Both SOD activities and their reactive oxidant forming correlated well with the copper ion content of the molecules. In addition, the proteins spontaneously aggregated and were precipitated by simple centrifugation at 12,000g for 20 min in keeping their enzyme activities. Since hyaline inclusions found in FALS patients with SOD1 mutations contained components which were reactive to anti-Cu,Zn-SOD antibody, a primary reaction caused by mutant SOD1 may be attributed to their propensity to form aggregates. Aggregated but still active mutant SOD1 would be expected to mediate the formation of reactive oxygen species and nitrosylation in a more condensed state.  相似文献   

18.
19.
Eight mutant Cu,Zn-superoxide dismutases (SODs) related to familial amyotrophic lateral sclerosis (FALS) were produced in a baculovirus/insect cell expression system and their molecular properties in terms of hydroxyl radical formation and aggregation were compared with the wild-type enzyme. Treatment of the enzymes with Chelex 100 resin decreased Cu contents as well as SOD activities in all mutant Cu,Zn-SODs, indicating that the affinities of the enzymes for copper ion were decreased. Contrary to previous reports, all the mutant Cu,Zn-SODs exhibited less reactive oxidant producing ability in the presence of hydrogen peroxide than the wild-type enzyme. Both SOD activities and their reactive oxidant forming correlated well with the copper ion content of the molecules. In addition, the proteins spontaneously aggregated and were precipitated by simple centrifugation at 12,000g for 20 min in keeping their enzyme activities. Since hyaline inclusions found in FALS patients with SOD1 mutations contained components which were reactive to anti-Cu,Zn-SOD antibody, a primary reaction caused by mutant SOD1 may be attributed to their propensity to form aggregates. Aggregated but still active mutant SOD1 would be expected to mediate the formation of reactive oxygen species and nitrosylation in a more condensed state.  相似文献   

20.
Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K(m) = 7.9 mM; k(cat)/K(m) = 0.73 mM(-1) s(-1)). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k(cat)/K(m) = 450 mM(-1) s(-1)) than with NADPH (k(cat)/K(m) = 5.5 mM(-1) s(-1)), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu(2+) and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.  相似文献   

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