Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.     

The single superoxide dismutase of Rhodobacter capsulatus is a cambialistic,manganese-containing enzyme

The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SOD(Rc)) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD(Rc), however, displayed higher activity after refolding with Mn(2+), especially when the pH of the assay mixture was raised. SOD(Rc) isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD(Rc) behaves as a Mn-containing dismutase with cambialistic properties.     

Superoxide dismutase 1 modulates expression of transferrin receptor

Copper–zinc superoxide dismutase (SOD1) plays a protective role against the toxicity of superoxide, and studies in Saccharomyces cerevisiae and in Drosophila have suggested an additional role for SOD1 in iron metabolism. We have studied the effect of the modulation of SOD1 levels on iron metabolism in a cultured human glial cell line and in a mouse motoneuronal cell line. We observed that levels of the transferrin receptor and the iron regulatory protein 1 were modulated in response to altered intracellular levels of superoxide dismutase activity, carried either by wild-type SOD1 or by an SOD-active amyotrophic lateral sclerosis (ALS) mutant enzyme, G93A-SOD1, but not by a superoxide dismutase inactive ALS mutant, H46R-SOD1. Ferritin expression was also increased by wild-type SOD1 overexpression, but not by mutant SOD1s. We propose that changes in superoxide levels due to alteration of SOD1 activity affect iron metabolism in glial and neuronal cells from higher eukaryotes and that this may be relevant to diseases of the nervous system.     

Therapeutic Effect of Liposomal Superoxide Dismutase in an Animal Model of Retinopathy of Prematurity

A newborn rat model of retinopathy of prematurity was used to test the hypothesis that a lack of superoxide dismutase contributes to the retinal vaso-attenuation seen during exposure of the animals to hyperoxic conditions. To determine the endogenous superoxide dismutase activity of the retina under hyperoxic conditions, litters of albino rats were placed in either constant 80% ambient oxygen (constant hyperoxia), or placed in 21% oxygen (room air) immediately after birth. Every other day, for 14 days, several rat pups were sacrificed and their retinas removed for the determination of total superoxide dismutase (SOD) activity and manganese-associated SOD activity. An attempt was made to increase retinal SOD activity by intraperitoneal administration of exogenous SOD encapsulated in polyethylene glycol-modified liposomes. Additional litters were exposed to the same oxygen treatments and supplemented twice daily with either liposome-encapsulated superoxide dismutase in saline or liposomes containing saline without SOD. Animals were sacrificed at various time points for the determination of total superoxide dismutase activity and computer-assisted analysis of vessel density and avascular area. Animals raised in an atmosphere of constant 80% oxygen had significantly reduced levels of retinal superoxide dismutase activity through 6 days of life when compared to their room air-raised littermates. At 6 days of age, daily supplementation with liposome-encapsulated SOD had significantly increased retinal superoxide dismutase activity and reduced oxygen-induced vaso-attenuation as evidenced by increased vessel density and decreased avascular area, when compared to littermates exposed to constant hyperoxia that received control liposomes. Superoxide dismutase had no adverse effects on any of the animals regardless of treatment. Tracing experiments demonstrated that liposomes entered the retina and were found in cells morphologically resembling mi-croglia. Delivery of SOD to the retina via long-circulating liposomes proved beneficial, suggesting that restoration and/or supplementation of endogenous antioxidants in oxygen-damaged retinal tissue is a potentially valuable therapeutic strategy.     

Regulation of superoxide dismutase synthesis in Candida albicans

The synthesis of superoxide dismutase [SOD: EC 1.15.1.1] in response to various cultural conditions was examined in Candida albicans, an opportunistic yeast which causes candidiasis in immunosuppressed patients. SOD plays an important role in protecting cells from the oxidative damage of superoxide radicals. Maximum SOD activity was found after 72 hrs of yeast growth. The optimum pH and temperature for the SOD activity were 7 and 40 °, respectively. The major SOD activity was found in the cytosol fraction and the level of extracellular SOD was very low. The enzyme was stimulated to varying degrees by cholic acid, procaine and tocopherol. On the basis of inhibitor studies and other enzyme properties, the isolated enzyme from C. albicans is identified as copper and zinc superoxide dismutase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Copper- and zinc-containing superoxide dismutase can act as a superoxide reductase and a superoxide oxidase

The copper- and zinc-containing superoxide dismutase can catalyze the oxidation of ferrocyanide by O(2) as well as the reduction of ferricyanide by O(2). Thus, it can act as a superoxide dismutase (SOD), a superoxide reductase (SOR), and a superoxide oxidase (SOO). The human manganese-containing SOD does not exert SOR or SOO activities with ferrocyanide or ferricyanide as the redox partners. It is possible that some biological reductants can take the place of ferrocyanide and can also interact with human manganese-containing superoxide dismutase, thus making the SOR activity a reality for both SODs. The consequences of this possibility vis à vis H(2)O(2) production, the overproduction of SODs, and the role of copper- and zinc-containing superoxide dismutase mutations in causing familial amyotrophic lateral sclerosis are discussed, as well as the likelihood that the biologically effective SOD mimics, as described to date, actually function as SORs.     

Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

Periplasmic superoxide dismutase from Desulfovibrio desulfuricans 1388 is an iron protein

It is shown that the genome of the sulfate-reducing bacterium Desulfovibrio desulfuricans 1388 contains a superoxide dismutase (SOD) gene (sod). The gene encodes an export signal peptide characteristic for periplasmic redox proteins. The amino acid sequence showed high homology with iron-containing SODs from other bacteria. Electrophoretically pure SOD was isolated from the periplasmic fraction of bacterial cells by FPLC chromatography. Like other Fe-SODs, D. desulfuricans 1388 superoxide dismutase is inhibited by H2O2 and azide, but not by cyanide.     

A novel,thermostable manganese-containing superoxide dismutase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

A new, thermostable superoxide dismutase (SOD) from Bacillus licheniformis M20, isolated from Bulgarian mineral springs, was purified 11-fold with 11% recovery of activity. From native PAGE and SDS-PAGE, the enzyme was composed of two subunits of 21.5 kDa each. The SOD was inhibited only by NaN₃, which suggested that this SOD is of the manganese superoxide dismutase type. The purified enzyme had maximum activity at pH 8 and 55°C. The half-life of the SOD was 10 min at 95°C.     

Somatic cell mapping of bovine EC-SOD and SOD1L loci.

cDNA probes of human extracellular superoxide dismutase (EC-SOD) and bovine superoxide dismutase 1 (SOD1) genes were hybridized to Southern blots containing genomic DNAs from cow-rodent somatic cell lines segregating bovine chromosomes. The SOD1 probe identified two loci: the coding locus (SOD1), which mapped to bovine U10; and a related locus (SOD1L), which mapped to U11. EC-SOD mapped to bovine U15. The mapping of EC-SOD to human chromosome 4, and our mapping of EC-SOD to U15, further defines a region of extensive syntenic conservation between humans and domestic cows.     

An estimate of the dipole moment of superoxide dismutase

A lower limit for the value of the dipole moment of superoxide dismutase (SOD) is calculated to be 485 Debye. This limit follows from the observation that the rate constant of the reaction between superoxide (O-2) and SOD decreases upon increasing the ionic strength, and the fact that at pH greater than 5 SOD has a net negative charge.     

Superoxide,hydrogen peroxide,and the gelling of phloem sap from Cucurbita pepo

The possible involvement of superoxide and hydrogen peroxide in the oxidative gelling of phloem exudate from Cucurbita pepo. was investigated. Neither superoxide dismutase (EC 1.15.1.1) nor catalase (EC 1.11.1.6) inhibited the reaction. Although catalase could not be detected in exudate, both peroxidase (EC. 1.11.1.7) and superoxide dismutase were present in reasonable amounts. Polyacrylamide gel electrophoresis revealed one major and one minor isozyme of superoxide dismutase, both of which were adjudged to contain copper and zinc as their prosthetic metals, on the basis of cyanide inhibition and molecular weight.Abbreviations SOD superoxide dismutase     

Growth of Chlorella sorokiniana in the presence of sulfite elevates cell content of superoxide dismutase and imparts resistance towards paraquat

1. Growth of Chlorella sorokiniana in the presence of 7.5 mM sulfite, which halved the growth rate while doubling the superoxide dismutase (SOD; EC 1.15.1.1) content per cell, rendered the cells resistant to the toxic effects of 30 M paraquat. 2. While increasing total SOD content, sulfite increased the relative amount of the H₂O₂-resistant manganese-containing SOD. 3. It appears that O₂ ^– may be involved in mediating the toxicity of SO₂ in this green alga.Abbreviations SOD superoxide, dismutase - FeSOD ironcontaining superoxide dismutase - MnSOD manganese-containing superoxide dismutase     

Chromatographic and electrophoretic methods for analysis of superoxide dismutases

A brief overview of the family of superoxide dismutase (SOD) enzymes and their biomedical significance is presented. Methodology for the purification and electrophoretic analysis of superoxide dismutases is reviewed and discussed, with emphasis on the specific problems raised by the separation of individual superoxide dismutase isoenzymes. Purification methods and their performance, as reported in the literature, are summarised in table form. Generally used methods for measuring SOD activity in vitro and SOD visualisation after electrophoresis are outlined, particularly those relevant to the monitoring of progress of SOD purification.     

Discovery of superoxide reductase: an historical perspective

For more than 30 years, the only enzymatic system known to catalyze the elimination of superoxide was superoxide dismutase, SOD. SOD has been found in almost all organisms living in the presence of oxygen, including some anaerobic bacteria, supporting the notion that superoxide is a key and general component of oxidative stress. Recently, a new concept in the field of the mechanisms of cellular defense against superoxide has emerged. It was discovered that elimination of superoxide in some anaerobic and microaerophilic bacteria could occur by reduction, a reaction catalyzed by a small metalloenzyme thus named superoxide reductase, SOR. Having played a major role in this discovery, we describe here how the concept of superoxide reduction emerged and how it was experimentally substantiated independently in our laboratory.Abbreviations Dfx desulfoferrodoxin - SOD superoxide dismutase - SOR superoxide reductase     

Cu,Zn superoxide dismutase increases intracellular calcium levels via a phospholipase C-protein kinase C pathway in SK-N-BE neuroblastoma cells

The superoxide dismutase isoenzymes (SOD) play a key role in scavenging, O*2- radicals. In contrast with previous studies, recent data have shown that human neuroblastoma cells are able to export the cytosolic Cu,Zn superoxide dismutase (SOD1), thus suggesting a paracrine role exerted by this enzyme in the nervous system. To evaluate whether SOD1 could activate intracellular signalling pathways, the functional interaction between SOD1 and human neuroblastoma SK-N-BE cells was investigated. By analyzing the surface binding of biotinylated SOD1 on SK-N-BE cells and by measuring intracellular calcium concentrations and PKC activity, we demonstrated that SOD1 specifically interacts in a dose-dependent manner with the cell surface membrane of SK-N-BE. This binding was able to activate a PLC-PKC-dependent pathway that increased intracellular calcium concentrations mainly deriving from the intracellular stores. Furthermore, we showed that this effect was independent of SOD1 dismutase activity and was totally inhibited by U73122, the PLC blocker. On the whole, these data indicate that SOD1 carries out a neuromodulatory role affecting calcium-dependent cellular functions.     

Manganese SOD Mimics Are Effective Against Heat Stress in a Mutant Fission Yeast Deficient in Mitochondrial Superoxide Dismutase

Previous studies revealed a close connection between heat shock and manganese-dependent superoxide dismutase (SOD2) in eukaryotes. This paper shows that SOD mimics based on manganese complexes caused an increase in thermotolerance for a mutant fission yeast deficient in mitochondrial superoxide dismutase. Manganese compounds used for tests are SOD mimics, from two different classes: salen manganese (EUK-8) and Mn porphyrin (Mn(III)TE-2-PyP(5+)). The tests were conducted using a Schizosaccharomyces pombe model, comparing the viability of two strains at chronic heat stress (37°C)--a wild type versus a strain with the mitochondrial superoxide dismutase gene deleted [SOD2(-)]. The presence of massive free radical species in S. pombe SOD2(-) was demonstrated using a luminol-enhanced chemiluminescence test derived from a menadione-mediated survival protocol. Conclusions: Survival tests revealed that the SOD2-deleted S. pombe is about 100 times more sensitive to heat stress than the wild-type strain. This survival deficit can be corrected by EUK-8 and Mn(III)TE-2-PyP(5+) to almost the same degree but not by manganese chloride II (MnCl(2)). Using a simple spot assay for viability testing, this new model proved to be an easy alternative for the initial estimation of manganese SOD mimics efficiency.     

Superoxide dismutase: fluctuations in the structure and solvation of the active site channel studied by molecular dynamics simulation

The molecular dynamics (MD) simulation of superoxide dismutase (SOD) in water is carried out for a total of 23 ps. The simulation system is a 26 A sphere centered at the active site of SOD, including 1602 atoms from SOD and 1761 water molecules. There is no gross deviation from the x-ray structure for the average MD structure. The structure and potential fluctuations around the active site are examined. The results provide new insight to the interactions between SOD and its substrate superoxide.     

Plasmodium falciparum: association with erythrocytic superoxide dismutase

Levels of superoxide dismutase (SOD) activity and its properties in Plasmodium falciparum-infected erythrocytes, isolated parasites, and noninfected erythrocytes were studied. A higher specific activity was found in P. falciparum-infected erythrocytes compared to noninfected erythrocytes, resulting from the lower protein content of infected cells and not enzyme synthesis by the parasite, as the superoxide dismutase activity expressed per number of cells was decreased. Superoxide dismutase from noninfected erythrocytes and isolated P. falciparum parasites showed similar sensitivities to various inhibitors and had identical molecular weights and electrophoretic mobilities. These results support the hypothesis of uptake and use of the erythrocytic SOD enzyme by the parasite as a possible mechanism of defense against oxidative stress.