Isoenzymes of cuprozinc superoxide dismutase from Pisum sativum

Two cyanide-sensitive and organic solvent-inactivated superoxide dismutase isoenzymes were purified from pea leaves, Pisum sativum, cv Thomas Laxto     

Extracellular superoxide dismutase attenuates release of pulmonary hyaluronan from the extracellular matrix following bleomycin exposure

The major pulmonary antioxidant enzyme involved in the protection of the lung interstitium from oxidative stress is extracellular superoxide dismutase (EC-SOD). It has been previously shown that EC-SOD knock-out mice are more susceptible to bleomycin-induced lung injury, however, the molecular mechanism(s) remains unclear. We report here that bleomycin-induced lung damage, in EC-SOD KO mice, is associated with increased hyaluronan release into alveolar fluid. Analysis of hyaluronan synthase gene expression and hyaluronan molecular weight distribution suggested that elevated levels of hyaluronan in the alveolar fluid are mostly due to its release from the interstitium. Our results indicate that EC-SOD attenuates bleomycin-induced pulmonary injury, at least in part, by preventing superoxide-mediated release of hyaluronan into alveolar space.     

Identification of superoxide dismutase isoenzymes in tobacco pollen

We investigated the possible existence of superoxide dismutase (SOD; EC 1.15.1.1) isoenzymes in the pollen of Nicotiana tabacum (Petit Havana SR-1 cultivar). To detect SOD activity, crude extracts from tobacco pollen were subjected to native polyacrylamide gel electrophoresis followed by staining with nitroblue tetra-zolium (NBT). The presence of six SOD isoenzymes was detected in tobacco pollen. Treatment with SOD inhibitors indicated the presence of one manganese SOD (Mn SOD), five copper-zinc SOD (Cu/Zn SOD) isoenzymes, and the absence of iron SOD (Fe SOD).     

Dissociation of superoxide production by mitochondrial complex I from NAD(P)H redox state

The relationship between the rate of superoxide production by complex I and NAD(P)H redox state was investigated in rat skeletal muscle mitochondria. A high rate of superoxide production was observed during succinate oxidation; the rate during pyruvate oxidation was over fourfold lower. However, the NAD(P)H pool was significantly less reduced during succinate oxidation than during pyruvate oxidation. We conclude that there is no unique relationship between superoxide production by complex I and the reduction state of the NAD(P)H pool. Our data suggest that less than 10% of the superoxide originates from the flavin site during reverse electron transport from succinate.     

Subcutaneous administration of bovine superoxide dismutase protects lungs from radiation-induced lung injury

Abstract

Background. The objective of the present study was to determine whether single administration of the antioxidant enzyme bovine superoxide dismutase (bSOD) after radiation therapy (RT) mitigates development of pulmonary toxicity in rats. Methods. Female F344 rats (n = 60) were divided among six experimental groups: (1) RT, single dose of 21 Gy to the right hemithorax; (2) RT + 5 mg/kg bSOD; (3) RT + 15 mg/kg bSOD; (4) No RT; (5) sham RT + 5 mg/kg bSOD; and (6) sham RT + 15 mg/kg bSOD. A single subcutaneous injection of bSOD (5 or 15 mg/kg) was administered 24 h post-radiation. The effects of bSOD on radiation-induced lung injury were assessed by measurement of body weight, breathing frequency, and histopathological changes. Immunohistochemistry was used to evaluate oxidative stress (8-OHdG⁺, NOX4⁺, nitrotyrosine⁺, and 4HNE⁺ cells), macrophage activation (ED1⁺), and expression of profibrotic transforming growth factor-β or TGF-β in irradiated tissue. Results. Radiation led to an increase in all the evaluated parameters. Treatment with 15 mg/kg bSOD significantly decreased levels of all the evaluated parameters including tissue damage and breathing frequency starting 6 weeks post-radiation. Animals treated with 5 mg/kg bSOD trended toward a suppression of radiation-induced lung damage but did not reach statistical significance. Conclusions. The single application of bSOD (15 mg/kg) ameliorates radiation-induced lung injury through suppression of reactive oxygen species/reactive nitrogen species or ROS/RNS-dependent tissue damage.     

Purification and characterization of Fe-containing superoxide dismutase from Methanobrevibacter arboriphilus strain AZ

Superoxide dismutase (SOD) was purified from cells of the strict anaerobic methanogenic archaeon Methanobrevibacter arboriphilus strain AZ. The four-step purification procedure resulted in enzyme with specific activity of 3970 units/mg and yield of 22%. It was shown that the SOD is a Fe-containing homotetramer composed of subunits of 21.2 kD each. Sodium azide (13.5 mM), unlike KCN, inhibits the activity of the SOD. Hydrogen peroxide (0.5 mM) inactivates the enzyme, which is consistent with the properties of the known Fe-containing SODs from methanogenic Archaea.     

Cu/Zn superoxide dismutase (VdSOD1) mediates reactive oxygen species detoxification and modulates virulence in Verticillium dahliae

The accumulation of reactive oxygen species (ROS) is a widespread defence mechanism in higher plants against pathogen attack and sometimes is the cause of cell death that facilitates attack by necrotrophic pathogens. Plant pathogens use superoxide dismutase (SOD) to scavenge ROS derived from their own metabolism or generated from host defence. The significance and roles of SODs in the vascular plant pathogen Verticillium dahliae are unclear. Our previous study showed a significant upregulation of Cu/Zn-SOD1 (VdSOD1) in cotton tissues following V. dahliae infection, suggesting that it may play a role in pathogen virulence. Here, we constructed VdSOD1 deletion mutants (ΔSOD1) and investigated its function in scavenging ROS and promoting pathogen virulence. ΔSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione. Despite lacking a signal peptide, assays in vitro by western blot and in vivo by confocal microscopy revealed that secretion of VdSOD1 is dependent on the Golgi reassembly stacking protein (VdGRASP). Both menadione-treated ΔSOD1 and cotton roots infected with ΔSOD1 accumulated more and less H₂O₂ than with the wildtype strain. The absence of a functioning VdSOD1 significantly reduced symptom severity and pathogen colonization in both cotton and Nicotiana benthamiana. VdSOD1 is nonessential for growth or viability of V. dahliae, but is involved in the detoxification of both intracellular ROS and host-generated extracellular ROS, and contributes significantly to virulence in V. dahliae.     

A new perspective on the role of CuZn superoxide dismutase (SOD1)

The CuZn superoxide dismutase (SOD1), a member of a group of isoenzymes involved in the scavenger of superoxide anions, is a dimeric carbohydrate free protein, mainly localized in the cytosol. The reactive oxygen species (ROS) are involved in many pathophysiological events correlated with mutagenesis, cancer, degenerative processes and aging. In the first part of this mini-review the well known role of SOD1 and ROS are briefly summarized. Following, a potential novel biological action that SOD1 could exert is described, based on the recent researches demonstrating the secretion of this enzyme in many cellular lines. Moreover, the role of impaired mutant SOD1 secretion, associated with cytoplasmic toxic inclusion, which occurs in familial amyotrophic lateral sclerosis (ALS), is summarized. In addition, a depolarization-dependent release of SOD1 in pituitary GH3 cells and in rat synaptosomes through a calcium and SNARE-dependent mechanism is reported.     

Regulation of superoxide dismutase isoforms in resistant and susceptible strawberry cultivars subjected to leaf spot disease

Abstract

Macroscopic symptoms were observed in two strawberry cultivars, with the degree of symptom intensity varying depending on the susceptibility of the cultivars, i.e. resistant or susceptible. The symptoms presented as red spots and were observed 30 d following leaf tissue inoculation with the Mycosphaerella fragariae pathogen. A comparison of the superoxide dismutase isoform profiles obtained by gel electrophoresis in all samples extracted from both resistant and susceptible cultivars indicated one constant sharp band, identified as Mn[sbnd]SOD with a molecular mass of 19 kDa. The intensity of this band was higher in all samples derived from the resistant cultivar than in those from the susceptible cultivar. Another superoxide dismutase (SOD) isoform, identified as CuZn[sbnd]SOD with a molecular mass of 16 kDa, was detected in all soluble proteins derived from the resistant cultivar. This isoform was not observed in the susceptible cultivar; however, following an incremental increase in the amount of loaded protein, it was illuminated as a faint band in a sample collected 3 d after inoculation, indicating insufficient production of the CuZn[sbnd]SOD isoform in the susceptible cultivar during an oxidative burst induced by the M. fragaria pathogen. Several bands were also characterized in both cultivars containing Fe and Mn as their co-factors (Fe, Mn[sbnd]SOD). Unlike in the resistant cultivar, where the activity of Fe, Mn[sbnd]SOD isoforms gradually and regularly increased and reached its highest level on the third day after inoculation, the activity of the isoforms changed irregularly over 20 days of study in the susceptible cultivar.     

哈茨木霉超氧化物歧化酶基因克隆与特性分析

构建了哈茨木霉菌丝的cDNA文库,并获得了3298条ESTs序列,对哈茨木霉(Trichoderma harzianum)ESTs序列本地数据库进行tBlastn检索,获得了哈茨木霉超氧化物歧化酶cDNA序列。cDNA序列全长751 bp,开放阅读框465bp,编码154个氨基酸组成的多肽,蛋白分子量为15.7kD。BlastP同源性分析表明该基因与麦角真菌(Claviceps purpurea)相似性最高为86%;与解脂耶氏酵母菌(Yarrowia lipolytica)相似性最低为72%。三级结构预测表明,其活性中心可能与His47,His49,His64,His72,His81,His121,D84位点有关,并构成其活性中心骨架。     

Effect of exercise on bone and articular cartilage in heterozygous manganese superoxide dismutase (SOD2) deficient mice

Abstract

Reactive oxygen species (ROS) are involved in both bone and cartilage physiology and play an important role in the pathogenesis of osteoporosis and osteoarthritis. The present study investigated the effect of running exercise on bone and cartilage in heterozygous manganese superoxide dismutase (SOD2)-deficient mice. It was hypothesized that exercise might induce an increased production of ROS in these tissues. Heterozygous SOD2-deficient mice should exhibit an impaired capability to compensate, resulting in an increased oxidative stress in cartilage and bone. Thirteen female wild type and 20 SOD2^+/? mice (aged 16 weeks) were randomly assigned to a non-active wild type (SOD2^+/+Con, n = 7), a trained wild type (SOD2^+/+Run, n = 6), a non-active SOD2^+/? (SOD2^+/?Con, n = 9) and a trained SOD2^+/? (SOD2^+/?Run, n = 11) group. Training groups underwent running exercise on a treadmill for 8 weeks. In SOD2^+/? mice elevated levels of 15-F_2t-isoprostane and nitrotyrosine were detected in bone and articular cartilage compared to wild type littermates. In osteocytes the elevated levels of these molecules were found to be reduced after exercise while in chondrocytes they were increased by aerobic running exercise. The observed changes in oxidative and nitrosative stress did neither affect morphological, structural nor mechanical properties of both tissues. These results demonstrate that exercise might protect bone against oxidative stress in heterozygous SOD2-deficient mice.     

Molecular evolution and structure--function relationships of the superoxide dismutase gene families in angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutases

This study assesses whether the phylogenetic relationships between SODs from different organisms could assist in elucidating the functional relationships among these enzymes from evolutionarily distinct species. Phylogenetic trees and intron positions were compared to determine the relationships among these enzymes. Alignment of Cu/ZnSOD amino acid sequences indicates high homology among plant sequences, with some features that distinguish chloroplastic from cytosolic Cu/ZnSODs. Among eukaryotes, the plant SODs group together. Alignment of the Mn and FeSOD amino acid sequences indicates a higher degree of homology within the group of MnSODs (>70%) than within FeSODs (approximately 60%). Tree topologies are similar and reflect the taxonomic classification of the corresponding species. Intron number and position in the Cu/Zn Sod genes are highly conserved in plants. Genes encoding cytosolic SODs have seven introns and genes encoding chloroplastic SODs have eight introns, except the chloroplastic maize Sod1, which has seven. In Mn Sod genes the number and position of introns are highly conserved among plant species, but not among nonplant species. The link between the phylogenetic relationships and SOD functions remains unclear. Our findings suggest that the 5' region of these genes played a pivotal role in the evolution of function of these enzymes. Nevertheless, the system of SODs is highly structured and it is critical to understand the physiological differences between the SODs in response to different stresses in order to compare their functions and evolutionary history.     

Deficiency of manganese superoxide dismutase in hepatocytes disrupts zonated gene expression in mouse liver

The liver acinus displays a physiological periportal to perivenous oxygen gradient. This gradient was implicated to use reactive oxygen species (ROS) as mediators for the zonal gene expression. Mitochondria use oxygen and produce ROS, therefore they may contribute to the zonation of gene expression. To further elucidate this, we used the Cre-loxP system to generate a hepatocyte-specific null mutation of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) in mice. We found that ROS levels were enhanced in livers of MnSOD(-/-) mice which were reduced in size and displayed signs of liver failure such as intracellular protein droplets, increased apoptotic bodies and Bax levels as well as multinuclear hepatocytes. Further, the zonation of glutamine synthetase, glucokinase and phosphoenolpyruvate carboxykinase was no longer preserved. We conclude that deficiency of mitochondrial MnSOD initiates a dysregulation of zonated gene expression in liver.     

Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS

Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of patients with familial amyotrophic lateral sclerosis (ALS). Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD's affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0-A-resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9 degrees twist of the subunits, which opens the SOD dimer interface and represents the largest intersubunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding subloop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 approximately 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may contribute to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations.     

Identification of aster yellows phytoplasma in garlic and green onion by PCR-based methods

In the summer of 1999, typical yellows-type symptoms were observed on garlic and green onion plants in a number of gardens and plots around Edmonton, Alberta, Canada. DNA was extracted from leaf tissues of evidently healthy and infected plants. DNA amplifications were conducted on these samples, using two primer pairs, R16F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. DNA samples of aster yellows (AY), lime witches'-broom (LWB) and potato witches'-broom (PWB) phytoplasmas served as controls and were used to determine group relatedness. In a direct polymerase chain reaction (PCR) assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected amplified products of 1.2 kb. Dilution (1/40) of each of the latter products were used as template and nested with specific primer pair R16(1)F1/R1. An expected PCR product of 1.1 kb was obtained from each phytoplasma-infected garlic and green onion samples, LWB and AY phytoplasmas but not from PWB phytoplasma. An aliquot from each amplification product (1.2 kb) with universal primers was subjected to PCR-based restriction fragment length polymorphism (RFLP) to identify phytoplasma isolates, using four restriction endonucleases (AluI, KpnI, MseI and RsaI). DNA amplification with specific primer pair R16(1)F1/R1 and RFLP analysis indicated the presence of AY phytoplasma in the infected garlic and green onion samples. These results suggest that AY phytoplasma in garlic and green onion samples belong to the subgroup 16Sr1-A.     

Enhancement of potency and stability of human extracellular superoxide dismutase

Cells express several antioxidant enzymes to scavenge reactive oxygen species (ROS) responsible for oxidative damages and various human diseases. Therefore, antioxidant enzymes are considered biomedicine candidates. Among them, extracellular superoxide dismutase (SOD3) had showed prominent efficacy against asthma and inflammation. Despite its advantages as a biomedicine, the difficulty in obtaining large quantity of active recombinant human SOD3 (rhSOD3) has limited its clinical applications. We found that a significant fraction of overexpressed rhSOD3 was composed of the inactive apo-enzyme and its potency against inflammation depended on the rate of metal incorporation. Also, purified rhSOD3 was unstable and lost its activity very quickly. Here, we suggest an ideal preparative method to express, purify, and store highly active rhSOD3. The enzymatic activity of rhSOD3 was maximized by incorporating metal ions into rhSOD3 after purification. Also, albumin or polyethylene glycol prevented rapid inactivation or degradation of rhSOD3 during preparative procedures and long-term storage. [BMB Reports 2015; 48(2): 91-96]