Purification,N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.     

Purification, N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.     

Cloning,production and characterisation of a recombinant Cu/Zn superoxide dismutase from Taenia solium

A full-length complementary DNA clone encoding a cytosolic Cu/Zn superoxide dismutase with a M(r) of 15,588 Da was isolated from a Taenia solium larvae complementary DNA library. Comparison analysis of its deduced amino acid sequence revealed a 71% identity with Schistosoma mansoni, 57.2-59.8% with mammalian and less than 54% with other helminth cytosolic Cu/Zn superoxide dismutase. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc enzymatic function are conserved. The T. solium Cu/Zn superoxide dismutase was expressed in the pRSET vector. Enzymatic and filtration chromatographic analysis showed a recombinant enzyme with an activity of 2,941 U/mg protein and a native M(r) of 37 kDa. Inhibition assays using KCN, H(2)O(2), NaN(3) and SDS indicated that Cu/Zn is the metallic cofactor in the enzyme. Thiabendazole (500 microM) and albendazole (300 microM) completely inhibited the activity of T. solium Cu/Zn superoxide dismutase. Thiabendazole had no effect on bovine Cu/Zn superoxide dismutase; in contrast, albendazole had a moderate effect on it at same concentrations. Antibodies against T. solium Cu/Zn superoxide dismutase did not affect the enzymatic function; nevertheless, it cross reacts with several Taenia species, but not with trematodes, nematodes, pig, human and bovine Cu/Zn superoxide dismutase enzymes. Western blot analysis indicated the enzyme was expressed in all stages. These results indicate that T. solium possesses a Cu/Zn superoxide dismutase enzyme that can protect him from oxidant-damage caused by the superoxide anion.     

Purification of a fully metal-depleted Cu, Zn superoxide dismutase from copper-deficient rat liver

A copper-deprived form of the enzyme Cu, Zn superoxide dismutase was identifiedin the liver of rats made copper-deficient by dietary restriction. In homogenates ofsuch livers Cu, Zn superoxide dismutase presents a dis-homogeneous electrophoreticprofile with respect to the native enzyme. When rat liver extracts were treated withexogenous copper an electrophoretic pattern resembling the native one was observed.Enzyme purified by chromatography on DE-52 resin shows two major components, onecorresponding to genuine, native enzyme and another one, eluting at higher ionicstrength. The latter protein (Fraction II) consists of several isoforms which showthe same characteristics of the native superoxide dismutase as far as immunoreactivityand molecular weight are concerned, but with decreased contents of copper and zinc. Itscatalytic constant, referring to copper content, was 15 times lower than that obtainedfor the native enzyme. Moreover, the catalytic power of purified Fraction II was notregained upon incubation with copper. The occurrence of a superoxide dismutase voidof metals confirms the hypothesis that this protein plays a dual physiological role:in metal metabolism and in superoxide anion dismutation.     

赤子爱胜蚓超氧化物歧化酶的纯化和部分性质研究

采用硫酸铵分级沉淀和柱层析的方法,从赤子爱胜蚓整体细胞抽提液内分离得到纯的铜锌超氧化物岐化酶（Ｃｕ,Ｚｎ－ＳＯＤ）。每１００ｇ蚯蚓得到的ＳＯＤ制品,总活力为１１１５０Ｕ,比活力为５１３８Ｕ／ｍｇ蛋白,回收率为２０％。铜锌超氧化物岐化酶呈淡蓝绿色,最大紫外吸收波长为２７０ｎｍ。测得该酶分子量为３３０００,亚基分子量为１６５００。该酶亚基由１５６个氨基酸残基组成,不含酪氨酸。     

Thermal stability and redox properties of M. tuberculosis CuSOD

The superoxide dismutase from Mycobacterium tuberculosis is the only Cu-containing superoxide dismutase that lacks zinc in the active site. To explore the structural properties of this unusual enzyme, we have investigated its stability by differential scanning calorimetry. We have found that the holo-enzyme is significantly more stable than the apo-protein or the partially metallated enzyme, but that its melting temperature is markedly lower than that of all the other characterized eukaryotic and prokaryotic Cu,Zn superoxide dismutases. We have also observed that, unlike the zinc-free eukaryotic or bacterial enzymes, the active site copper of the mycobacterial enzyme is not reduced by ascorbate, confirming that its redox properties are comparable to those typical of the enzymes containing zinc in the active site. Our findings highlight the role of zinc in conferring stability to Cu,Zn superoxide dismutases and indicate that the structural rearrangements observed in M. tuberculosis Cu,SOD compensate for the absence of zinc in achieving a fully active enzyme.     

狗肝铜锌超氧化物歧化酶的纯化

本文采用加热、硫酸铵分级沉淀和柱层析的方法,从狗肝中提纯了铜锌超氧化物歧化酶(Cu·Zn-SOD),并对其理化性质进行了鉴定。结果表明酶的纯度均一。与文献报道的不同来源的同类酶相同,狗肝Cu·Zn-SOD系由两个相同亚基组成的二聚体,每分子晦蛋白合有两个铜和两个锌原子。分子量33.6kD,N-末端氨基酸为丙氨酸。     

重组人超氧化物歧化酶化学修饰的初步研究

在高效表达重组人铜锌超氧化物歧化酶(rh Cu/Zn SOD),并纯化得到比活大于4000单位的 rh Cu/Zn SOD 纯品的基础上,采用活化酯法将聚乙二醇(PEG)与 rhCu/Zn SOD 交联,获得分子量约6万的 PEG-SOD 交联物.经 PEG 修饰的酶稳定性增强,表现为对酸、碱和热的耐受力均较未交联酶高.修饰酶的生物半衰期为15h,是天然酶的90倍,酶活性保留80%以上.还实验观察了修饰剂用量与修饰酶保留活性之间的关系.     

Mechanism and Atomic Structure of Superoxide Dismutase

The active site Cu ion in Cu,Zn superoxide dismutase is alternately oxidized and reduced during the enzymatic dismutation of superoxide to hydrogen peroxide and molecular oxygen. For oxidized Cu,Zn superoxide dismutase, an atomic structure has been determined for the human enzyme at 2.5 A resolution. The resolution of the bovine enzyme structure has been extended to 1.8 A. Atomic resolution data has been, collected for reduced and inhibitor-bound Cu,Zn superoxide dismutases. and the interpretation of the' electron density difference maps is in progress. The geometry and molecular surfaces of the active sites in these structures, together with biochemical data, suggest a specific model for the enzyme mechanism. Similarities in the active site geometry of the Mn and Fe superoxide dismutases with the Cu.Zn enzyme suggest that dismutation in these enzymes may follow a similar mechanism.     

Purification and Characterization of Cu,Zn Superoxide Dismutase from Ark Shell Scapharca broughtonii

A superoxide dismutase has been purified to apparent homogeneity from the muscular tissue of the ark shell, Scapharca broughtonii, by ammonium sulfate fractionation, and consecutive column chromatographies using DEAE-Sephadex and Sephadex G-100. This enzyme has a molecular weight of 71,700 and is composed of two identical subunits of M _r 35,800, which are joined by noncovalent interactions. The purified enzyme was stable over the range of pH 5.0-10.0 at 4°C for 24 h and at temperatures below 45°C. Cyanide at 0.1 and 1 mM inhibited the activity of the superoxide dismutase 56 and 100%, but 5 mM azide caused 8% inhibition. The optical spectrum of this enzyme had a maximum at 265 nm, and the amino acid composition of the enzyme was similar to that of the other Cu, Zn superoxide dismutases except for the contents of threonine, serine, proline, and leucine. Atomic absorption spectroscopy showed that this enzyme has approximately 2 atoms of Cu²⁺ and Zn²⁺ per mole of enzyme. These results indicate that the purified enzyme from ark shell, Scapharca broughtonii, is a Cu, Zn superoxide dismutase.     

Role of the electrostatic loop charged residues in Cu,Zn superoxide dismutase.

We have expressed and characterized a mutant of Xenopus laevis Cu,Zn superoxide dismutase in which four highly conserved charged residues belonging to the electrostatic loop have been replaced by neutral side chains: Lys120 --> Leu, Asp130 --> Gln, Glu131 --> Gln, and Lys134 --> Thr. At low ionic strength, the mutant enzyme is one of the fastest superoxide dismutases ever assayed (k = 6.7 x 10(9) M(-1) s(-1), at pH 7 and mu = 0.02 M). Brownian dynamics simulations give rise to identical enzyme-substrate association rates for both wild-type and mutant enzymes, ruling out the possibility that enhancement of the activity is due to pure electrostatic factors. Comparative analysis of the experimental catalytic rate of the quadruple and single mutants reveals the nonadditivity of the mutation effects, indicating that the hyperefficiency of the mutant is due to a decrease of the energy barrier and/or to an alternative pathway for the diffusion of superoxide within the active site channel. At physiological ionic strength the catalytic rate of the mutant at neutral pH is similar to that of the wild-type enzyme as it is to the catalytic rate pH dependence. Moreover, mutation effects are additive. These results show that, at physiological salt conditions, electrostatic loop charged residues do not influence the diffusion pathway of the substrate and, if concomitantly neutralized, are not essential for high catalytic efficiency of the enzyme, pointing out the role of the metal cluster and of the invariant Arg141 in determining the local electrostatic forces facilitating the diffusion of the substrate towards the active site.     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

Antigenicity of filarial superoxide dismutase in human bancroftian filariasis

Superoxide dismutase activity was measured in different stages of growth of filarial parasites (human and cattle). The activity was almost undetected or very low in microfilarial stage but in adult worms, the enzyme activity was high. The enzyme was characterized to be a Cu/Zn superoxide dismutase. Most of the enzyme activity was associated with a detergent extractable fraction of adult (Setaria) parasite. The enzyme was also detected in thein vitro released products of adult worms. The superoxide dismutase activity was completely inhibited with IgG antibody from chronic filarial patients in contrast to IgG from normal people. Filarial patients particularly have high IgG and IgM antibody levels to purified enzyme. However, individuals from non-filarial regions of Orissa are sero-negative for superoxide dismutase antibodies. Antibody response to superoxide dismutase could thus be used for filarial diagnosis.     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

人铜锌超氧化物歧化酶cDNA的克隆,测序及表达

用逆转录聚合酶链反应（ＲＴＰＣＲ）,以人胎肝组织总ＲＮＡ为模板,扩增了人铜锌超氧化物歧化酶（ｈＣｕ,ＺｎＳＯＤ）的ｃＤＮＡ,并进行序列分析,将该ｈＣｕ,ＺｎＳＯＤｃＤＮＡ重组到Ｔ７启动子控制下的分泌型表达载体ｐＥＴ２２ｂ（＋）中,构建表达质粒ｐＥＴＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代βＤ半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为１９ｋＤ的蛋白质,与抗人ＳＯＤ多抗有特异的免疫反应,表达量约为菌体总蛋白质的３０％,具有特异性ＳＯＤ酶活性,酶活力可达１７９７ｕ／ｍｌ培基。     

Probing the Structural Basis for Enzyme-Substrate Recognition In Cu,Zn Superoxide Dismutase

A full understanding of enzyme-substrate interactions requires a detailed knowledge of their structural basis at atomic resolution. Crystallographic and biochemical data have been analyzed with coupled computational and computer graphic approaches to characterize the molecular basis for recognition of the superoxide anion substrate by Cu. Zn superoxide dismutase (SOD). Detailed analysis of the bovine SOD structure aligned with SOD sequences from 15 species provides new results concerning the significance and molecular basis for sequence conservation. Specific roles have been assigned for all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stcreochemistry. supporting a primary biological function of superoxide dismutation. Using data from crystallographic structures and site-directed mutants, we are testing the role of individual residues in the active site channel, including (in human SOD) Glu132, Glu133, Lys136, Thr137, and Arg 143. Electrostatic calculations incorporating molecular flexibility suggest that the region of positive electrostatic potential in and over the active site channel above the Cu ion sweeps through space during molecular motion to enhance the facilitated diffusion responsible for the enzyme's rapid catalytic rate.     

Purification,Characterization, and Molecular Cloning of a Thermostable Superoxide Dismutase from Thermoascus aurantiacus

A thermostable superoxide dismutase [(SOD) EC 1.15.1.1] from a Thermoascus aurantiacus var. levisporus was purified to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) homogeneity by a series of column chromatographies. The molecular mass of a single band of the enzyme was estimated to be 16.8 kDa by SDS–PAGE. The molecular mass was estimated to be 33.2 kDa by gel filtration on Sephacryl S-100, indicating that the enzyme was composed of two identical subunits of 16.8 kDa each. N-terminal amino acid sequencing (seven residues) yielded VKAVAVL. Using RACE-PCR, a Cu, Zn-SOD gene was cloned from T. aurantiacus var. levisporus. The sequence was 705 bp and contained a 468 bp ORF encoding a Cu, Zn-SOD of 155 amino acid residues.     

A proposal for the metal geometry in yeast superoxide dismutase based on results from EXAFS spectroscopy

Extended x-ray absorption fine structure (EXAFS) spectra have been recorded at the Cu edge and Zn edge in native yeast superoxide dismutase and at the Cu edge and Cd edge in the yeast superoxide dismutase derivative, where Zn has been substituted with Cd. Two different metal ligand distances in the range 1.9-2.0 A and 2.3-2.4 are determined for the Cu and Zn metals. For Cd in the Zn site two different metal ligand distances about 2.2 A and 2.6 A, respectively, were found. The striking feature is the similarity between the amplitude and radii determined for both the Cu and Zn sites. The increased distances for Cd can be explained by the increased ionic radius of Cd relative to Cu and Zn. Based on these EXAFS results and other relevant knowledge about the metal geometries, we propose that histidine 61 (63) positioned between the Cu and Zn metals are in one subunit bound to Zn and in the other to Cu. This model explains the recently observed difference between the two metal sites in each subunit.     

Characterization of Fish Cu/Zn–Superoxide Dismutase and Its Protection from Oxidative Stress

Copper/zinc superoxide dismutase was cloned from the zebrafish (Danio rerio). The full coding region of the zebrafish superoxide dismutase (ZSOD) complementary DNA was ligated with pET-20b(+) and successfully expressed in Escherichia coli strain AD494(DE3)pLysS. The active enzyme was purified by His tagging. The ZSOD yield was 6 mg from 0.2 L of E. coli culture, and the specific activity was 2000 U/mg as assayed using a RANSOD kit. The enzyme stability was characterized by reaction to temperature, pH, and detergent treatment. The results showed enzyme activity was still active after heat treatment at 70°C for 10 minutes, resistant to pH treatment from 2.3 to 12, and resistant to treatment with sodium dodecyl sulfate (SDS) under 4%. In addition, the recombinant ZSOD was used to protect fish from 100 ppm of paraquat-induced oxidative injury by soaking fish larva in 55 µg/ml SOD enzyme. The results were significant.     

Superoxide dismutase isoenzymes in bovine and human milk

The presence of superoxide dismutase in bovine and human milk was investigated by ultrafiltration, gel filtration, and isoelectric focusing. Conclusive evidence for the presence of this enzyme in both milks is presented. The molecular weight of the enzyme was estimated by gel filtration on Sephadex G-100 to be 30,000, which is consistent with reported values for the copper, zinc form of superoxide dismutase. In addition, enzyme activity was inhibited by cyanide, thus eliminating the possibility that the enzyme was present in the manganese form. Several isoenzymes were detected by isoelectric focusing in polyacrylamide gel, and the isoenzyme pattern in bovine milk was the same as that found for bovine plasma, suggesting that milk superoxide dismutase originates from plasma. It may be that the presence of copper, zinc superoxide dismutase in milk is important for the maintenance of its oxidative stability.