Use of cyanide and diethyldithiocarbamate in the assay of superoxide dismutases.

Eucaryotes have two major forms of superoxide dismutase (SOD), Cu,ZnSOD and MnSOD; in most tissues Cu,ZnSOD is present in higher amounts than MnSOD. To assay MnSOD, Cu,ZnSOD can be inhibited selectively by millimolar concentrations of cyanide ion. However, calculation of MnSOD activity from the differential cyanide inhibition assay is complex and small experimental errors can cause large errors in the calculated MnSOD activity. We have assessed how interaction of cyanide and hydrogen peroxide with cytochrome c can lead to further errors in the xanthine oxidase-cytochrome c assay for SOD. Alternatively, Cu,ZnSOD can be completely inactivated by 50 mM diethyldithiocarbamate (DDC) at 30 degrees C for 1 h without affecting the activity of MnSOD. Since DDC reduces cytochrome c, the treated samples must be thoroughly dialyzed or desalted before assay. In the case of lung homogenates, dialysis is not an extra step since fresh, untreated samples must also be dialyzed or desalted before assaying by the cytochrome c method. Cu,ZnSOD activity is equal to the activity in the untreated sample minus the activity in the DDC-treated portion of the sample. Another copper chelator, triethylenetetramine, did not inactivate Cu,ZnSOD and could not be used instead of DDC. For accurate measurement of both enzymes in samples where MnSOD contributes only a small fraction of the total SOD activity, the DDC method has the advantage that it provides a direct measure of the MnSOD activity without interference by Cu,ZnSOD.     

Expression of superoxide dismutases in the bovine oviduct during the estrous cycle

The superoxide dismutases (SODs) are first-line enzymatic antioxidants that dismute superoxide anion (O(2)(-)) to produce hydrogen peroxide (H(2)O(2)). The primary objective was to characterize, by western blot analysis, the expression of two SODs, the cytosolic (Cu,ZnSOD or SOD1) and the mitochondrial (MnSOD or SOD2) forms in three sections of the oviduct, i.e. isthmus (I), ishtmic-ampullary junction (IA), and ampulla (A), during the estrous cycle. The Cu,ZnSOD and MnSOD proteins were mostly expressed in the ampulla (I相似文献   

Cryo-trapping the six-coordinate, distorted-octahedral active site of manganese superoxide dismutase

Superoxide dismutase protects organisms from potentially damaging oxygen radicals by catalyzing the disproportionation of superoxide to oxygen and hydrogen peroxide. We report the use of cryogenic temperatures to kinetically capture the sixth ligand bound to the active site of manganese superoxide dismutase (MnSOD). Synchrotron X-ray diffraction data was collected from Escherichia coli MnSOD crystals grown at pH 8.5 and cryocooled to 100 K. Structural refinement to 1.55 A resolution and close inspection of the active site revealed electron density for a sixth ligand that was interpreted to be a hydroxide ligand. The six-coordinate, distorted-octahedral geometry assumed during inhibition by hydroxide is compared to the room temperature, five-coordinate, trigonal bipyramidal active site determined with crystals grown from practically identical conditions. The gateway residues Tyr34, His30 and a tightly bound water molecule are implicated in closing-off the active site and blocking the escape route of the sixth ligand.     

锰超氧化物歧化酶的催化原理与酶活性调节机制

锰超氧化物歧化酶(MnSOD)催化两分子超氧自由基歧化为分子氧和过氧化氢。超氧自由基被Mn³⁺SOD氧化成分子氧的反应以扩散的方式进行。超氧自由基被Mn²⁺SOD还原为过氧化氢的反应以快循环和慢循环两条途径平行进行。在慢循环途径中,Mn²⁺SOD与超氧自由基形成产物抑制复合物,然后该复合物被质子化而缓慢释放出过氧化氢。在快循环途径中,超氧自由基直接被Mn²⁺SOD转化为产物过氧化氢,快速循环有利于酶的复活与周转。本文提出温度是调节锰超氧化物歧化酶进入慢速或者快速循环催化途径的关键因素。随着在生理温度范围内的温度升高,慢速循环成为整个催化反应的主流,因而生理范围内的温度升高反而抑制该酶的活性。锰超氧化物歧化酶的双相酶促动力学特性可以用该酶保守活性中心的温度依赖性配位模型进行合理化解释。当温度降低时,1个水分子(或者OH^-)接近Mn、甚至与Mn形成配位键,从而干扰超氧自由基与Mn形成配位键而避免形成产物抑制。因此在低温下该酶促反应主要在快循环通路中进行。最后阐述了几种化学修饰模式对...     

Antioxidant response system in the short-term post-wounding effect in Mesembryanthemum crystallinum leaves

Mechanical wounding of Mesembryanthemum crystallinum leaves in planta induced a fast decrease in stomatal conductance, which was related to accumulation of hydrogen peroxide (H(2)O(2)). Higher levels of H(2)O(2) were accompanied by an increase in total activity of superoxide dismutase (SOD) and a decrease in catalase (CAT) activity. Among SOD forms, manganese SOD (MnSOD) and copper/zinc SOD (Cu/ZnSOD) seem to be especially important sources of H(2)O(2) at early stages of wounding response. Moreover, NADP-malic enzyme (NADP-ME), one of the key enzymes of primary carbon metabolism, which is also involved in stress responses, showed a strong increase in activity in wounded leaves. All these symptoms: high accumulation of H(2)O(2), high activities of Cu/ZnSOD and NADP-ME, together with the decrease of CAT activity, were also observed in the major veins of unwounded leaves. The potential role of veinal tissues as an important source of H(2)O(2) during wounding response is discussed.     

Catalytic and structural effects of amino acid substitution at histidine 30 in human manganese superoxide dismutase: insertion of valine C gamma into the substrate access channel

Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD) is characterized by an initial burst of catalysis followed by a much slower region that is zero order in superoxide and due to a product inhibition by peroxide anion. We have prepared site-specific mutants with replacements at His30, the side chain of which lies along the substrate access channel and is about 5.8 A from the metal. Using pulse radiolysis to generate superoxide, we have determined that kcat/K(m) was decreased and product inhibition increased for H30V MnSOD, both by 1-2 orders of magnitude, compared with wild type, H30N, and H30Q MnSOD. These effects are not attributed to the redox potentials, which are similar for all of these variants. An investigation of the crystal structure of H30V Mn(III)SOD compared with wild type, H30Q, and H30N Mn(III)SOD showed the positions of two gamma carbons of Val30 in the active site; Cgamma1 overlaps Cgamma of His30 in wild type, and Cgamma2 extends into the substrate access channel and occupies the approximate position of a water molecule in the wild type. The data suggest that Cgamma2 of the Val side chain has significantly interrupted catalysis by this overlap into the access channel with possible overlap with the substrate-product binding site. This is supported by comparison of the crystal structure of H30V MnSOD with that of azide bound to Mn(III)SOD from Thermus thermophilus and by visible absorption spectra showing that azide binding to the metal in H30V Mn(III)SOD is abolished. Moreover, the presence of Val30 caused a 100-fold decrease in the rate constant for dissociation of the product-inhibited complex compared with wild type.     

Micronutrient Deficiency Influences Plant Growth and Activities of Superoxide Dismutases in Narrow-leafed Lupins

The effect of copper (Cu), zinc (Zn) or manganese (Mn) deficiencyon the growth and activity of superoxide dismutase (SOD) formswas investigated in seedlings of narrow-leafed lupins (LupinusangustifoliusL.). Plants grown without Zn developed Zn deficiencysymptoms 24 d after sowing (DAS), and those grown without Mnshowed Mn deficiency symptoms 31 DAS. However, plants grownwithout Cu did not show visible leaf symptoms. Shoot dry weightwas decreased by Zn and Mn deficiency 24 DAS, and by Cu deficiency31 DAS. Soluble protein concentration was reduced considerablyby Zn deficiency 24 DAS, but was not affected by Cu deficiencyuntil 31 DAS. In contrast, soluble protein concentration inMn-deficient plants was higher than in control plants 31 DAS.Shoot concentration of micronutrients which were not suppliedto plants decreased significantly, with a simultaneous increasein concentration of one or more of the other nutrients analysed.The activities of total SOD, MnSOD and Cu/ZnSOD on a fresh weightbasis declined drastically in -Cu and -Zn plants 24 DAS. Onthe contrary, the activities of total SOD and Cu/ZnSOD on eithera fresh weight or soluble protein basis increased markedly in-Mn plants 24 DAS, and MnSOD activity increased significantlyin these plants 31 DAS. It was concluded that micronutrientdeficiency (Cu, Zn or Mn) altered the activities of SOD formsdepending on the kind and severity of the deficiency stress.Manipulation of the capacity of plants to tolerate oxidativestress may influence their capacity to tolerate micronutrientdeficiency.Copyright 1999 Annals of Botany Company. Copper,Lupinus angustifolius, manganese, deficiency, superoxide dismutase, zinc.     

Superoxide dismutase activity in salt stressed wheat seedlings

We investigated the effect of salt stress on enzymatic activity of superoxide dismutase (SOD) isozymes in shoot and root tissues of salt tolerant and sensitive wheat (Triticum aestivum L. and Triticum durum Defs.) cultivars. Ten day old seedlings were subjected to 0.7 M NaCl stress for 3 and 5 days. Seedlings treated in the same manner without salt stress served as controls. Activity of SOD isozymes in root and shoot extracts was determined by activity staining of native polyacrylamide gels. In both shoot and root extracts of examined cultivars two isozymes of SOD, namely MnSOD and Cu/ZnSOD were identified. Cu/ZnSOD activity comprised 90 % of total SOD activity in both root and shoot tissues. Salt stress caused 1–1.5 fold increase in MnSOD activity of shoots in tolerant cultivars when compared with non-stressed controls. Under stress conditions, compared to controls all cultivars exhibited reduced MnSOD activity in root tissues. Cu/ZnSOD activity, on the other hand, was remarkably enhanced (3–4 fold) in root extracts of the tolerant cultivars, whereas it was reduced in the sensitive ones.     

The structural biochemistry of the superoxide dismutases

The discovery of superoxide dismutases (SODs), which convert superoxide radicals to molecular oxygen and hydrogen peroxide, has been termed the most important discovery of modern biology never to win a Nobel Prize. Here, we review the reasons this discovery has been underappreciated, as well as discuss the robust results supporting its premier biological importance and utility for current research. We highlight our understanding of SOD function gained through structural biology analyses, which reveal important hydrogen-bonding schemes and metal-binding motifs. These structural features create remarkable enzymes that promote catalysis at faster than diffusion-limited rates by using electrostatic guidance. These architectures additionally alter the redox potential of the active site metal center to a range suitable for the superoxide disproportionation reaction and protect against inhibition of catalysis by molecules such as phosphate. SOD structures may also control their enzymatic activity through product inhibition; manipulation of these product inhibition levels has the potential to generate therapeutic forms of SOD. Markedly, structural destabilization of the SOD architecture can lead to disease, as mutations in Cu,ZnSOD may result in familial amyotrophic lateral sclerosis, a relatively common, rapidly progressing and fatal neurodegenerative disorder. We describe our current understanding of how these Cu,ZnSOD mutations may lead to aggregation/fibril formation, as a detailed understanding of these mechanisms provides new avenues for the development of therapeutics against this so far untreatable neurodegenerative pathology.     

The structure of iron superoxide dismutase from Pseudomonas ovalis complexed with the inhibitor azide

The 2.9 A resolution structure of iron superoxide dismutase (FeSOD) (EC 1.15.1.1) from Pseudomonas ovalis complexed with the inhibitor azide was solved. Comparison of this structure with free enzyme shows that the inhibitor is bound at the open coordination position of the iron, with a bond length of 2.0 A. The metal moves by 0.4 A into the trigonal plane to produce an orthogonal geometry at the iron. Binding of the inhibitor also causes a movement of the axial ligand (histidine 26) away from the metal, a lengthening of the iron-histidine bond, and a rotation of the histidine 74 ring. The inhibitor possesses contacts in the binding pocket with a pair of conserved tryptophan residues and with the side chains of tyrosine 34 and glutamine 70. This glutamine is conserved between all FeSODs, but is absent in MnSOD. Comparisons with MnSOD show that a different glutamine which possesses the same interactions in the active site as Gln70 in FeSOD is conserved at position 154 in the overall SOD sequence, implying that while manganese and FeSODs are structural homologues in a global sense, their functional and evolutionary relationship is that of second-site mutation revertants.     

Protection of insulin-producing cells against toxicity of dexamethasone by catalase overexpression

Pancreatic β cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in β cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic β cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis.     

DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O _inf2 ^sup– , to O₂ and H₂O₂, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may he injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H₂O₂. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.     

Recombinant Human Superoxide Dismutases: Production and Potential Therapeutical Uses

In many pathological situations, tissue damage is caused by cellular generation of superoxide free radicals (O₂-). These active species are generated during post-ischemic reperfusion of organs, in hyperoxic tissue, during acute and chronic inflammation and during exposure to ionizing radiation. Exogenous superoxide dismutase (SOD) was shown to significantly prevent such damage.

The genes for human cytosolic Cu/ZnSOD and mitochondrial MnSOD were cloned and introduced into an E. coli expression system. The proteins were expressed in high yields and purified to homogeneity, yielding pharmaceutical-grade materials. These enzymes were used in a variety of in vivo animal models for the demonstration of their protective effects against oxidative damage. Comparative pharmacokinetic studies in rats have revealed that the half-life of Cu/ZnSOD was 6–10min., while that of MnSOD was 5–6 hours, thus indicating that MnSOD may be superior to Cu/ZnSOD for the treatment of chronic diseases. Indeed, MnSOD was found to be erective as an anti-inflammatory agent in the rat carrageenan induced paw edema acute inflammation model. Both enzymes were also effective in ameliorating post-irradiation damage in mice exposed to whole-body or localized chest X-ray radiation.     

Crystallographic determination of reduced bovine superoxide dismutase at pH 5.0 and of anion binding to its active site

The crystal structures of dithionite-reduced bovine Cu(I),Zn superoxide dismutase and of its adducts with the inorganic anions azide and thyocyanide have been determined in a C222₁ crystal form obtained at pH?5.0. This crystal form is characterized by a high solvent content (72%) and by having the two Cu,ZnSOD monomers (A and B) in different crystal environments. One of them (B) is involved in few intermolecular crystal contacts so that it is in a more "solution like" environment, as indicated by average temperature factors which are about twice those of the other monomer. The differences in crystal packing affect the active site structures. While in the A monomer the Cu(I) is coordinated to all four histidine residues, in the B monomer the bridging His61 side chain is found disordered, implying partial detachment from copper. The same effect occurs in the structures of the anion complexes. The inorganic anions are found bound in the active site cavity, weakly interacting with copper at distances ranging from 2.5 to 2.8?Å. The copper site in the A subunit of the native enzyme structure displays significant electron density resembling a diatomic molecule, bound side-on at about 2.8?Å from the metal, which cannot be unambiguously interpreted. The crystallographic data suggest that the existence of the His61 bridge between copper and zinc is dominated by steric more than electronic factors and that the solution state favors the His61 detachment. These structures confirm the existence of an energetically available state for Cu(I) in Cu,ZnSOD where the histidinato bridge to zinc is maintained. This state appears to be favored by tighter crystal contacts. The binding of the anions in the active site cavity is different from that observed in the oxidized enzyme and it appears to be dominated by electrostatic interactions within the cavity. The anion binding mode observed may model the substrate interaction with the reduced enzyme during catalysis.     

Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities

Germin is a hydrogen peroxide generating oxalate oxidase with extreme thermal stability; it is involved in the defense against biotic and abiotic stress in plants. The structure, determined at 1.6 A resolution, comprises beta-jellyroll monomers locked into a homohexamer (a trimer of dimers), with extensive surface burial accounting for its remarkable stability. The germin dimer is structurally equivalent to the monomer of the 7S seed storage proteins (vicilins), indicating evolution from a common ancestral protein. A single manganese ion is bound per germin monomer by ligands similar to those of manganese superoxide dismutase (MnSOD). Germin is also shown to have SOD activity and we propose that the defense against extracellular superoxide radicals is an important additional role for germin and related proteins.     

The 2.0A resolution structure of the catalytic portion of a cyanobacterial membrane-bound manganese superoxide dismutase

Cyanobacteria are shown to be unique in containing membrane-bound manganese superoxide dismutases (MnSOD). They are homodimeric type 2 membrane proteins that protect this phototrophic organism against oxidative stress. We have determined, for the first time, the 2.0A resolution structure of the catalytic portion of the MnSOD from the filamentous cyanobacterium Anabaena PCC 7120. Within each subunit, both the N-terminal helical hairpin (His94 and His145) and the C-terminal alpha/beta domain (His232 and Asp228) contribute ligands to the catalytic manganese site. Together with a water or hydroxide ion (OH(x)) a five-coordinated trigonal bipyramidal geometry is formed, with OH(x) and His90 forming the axial ligands and manganese shifted out of the equatorial plane in the direction of OH(x). The ligands including OH(x) are tightly constrained by hydrogen bonding with surrounding residues either from the same monomer (Tyr98, Asn144, Trp194, Gln213, Val229, Trp230) or from the neighbouring subunit (Glu231, Tyr235). This underlines the important role of the symmetric dimeric structure of MnSODs in contributing elements to both the active site and the substrate funnel. The Mn cdots, three dots, centered Mn distance (18.4A) is bridged by the hydrogen-bonded His232 of one monomer with Glu231 of the other monomer. A detailed discussion of the structure, a comparison with known structures of soluble MnSODs as well as a model of the cyanobacterial membrane-bound MnSOD is presented.     

Characterization of the Bacillus stearothermophilus manganese superoxide dismutase gene and its ability to complement copper/zinc superoxide dismutase deficiency in Saccharomyces cerevisiae.

Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could function in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomyces cerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol.     

Effect of hypoxia on photosynthetic activity and antioxidative response in gametophores of <Emphasis Type="Italic">Mnium undulatum</Emphasis>

The effects of hypoxia caused by complete submerging of Mnium undulatum gametophores in water, on their photosynthetic activity and the activity of two antioxidative enzymes: superoxide dismutase (SOD) and catalase (CAT) were investigated. The net photosynthesis was strongly inhibited throughout the experiment, and the strong drop in the maximum quantum yield of the PSII (F_v/F_m) was also observed. Three classes of SOD: MnSOD, FeSOD, Cu/ZnSOD and three isoforms of Cu/ZnSOD were identified. A significant decrease in activity of MnSOD, FeSOD and one Cu/ZnSOD isoform was observed after 24 and 48 h of hypoxia. FeSOD activity decreased already after 1 h of submerging in water and its activity remained at the low level during whole period of the experiment. CAT activity was also strongly inhibited in response to hypoxia stress. The obtained results suggest relationships between photosynthetic activity and antioxidative system in M. undulatum gametophores under oxygen deficiency stress.     

Kinetic analysis of product inhibition in human manganese superoxide dismutase

Manganese superoxide dismutase (MnSOD) cycles between the Mn(II) and Mn(III) states during the catalyzed disproportionation of O(2)(*-), a catalysis that is limited at micromolar levels of superoxide by a peroxide-inhibited complex with the metal. We have investigated the role in catalysis and inhibition of the conserved residue Trp161 which forms a hydrophobic side of the active site cavity of MnSOD. Crystal structures of mutants of human MnSOD in which Trp161 was replaced with Ala or Phe showed significant conformational changes on adjacent residues near the active site, particularly Gln143 and Tyr34 which in wild-type MnSOD participate in a hydrogen bond network believed to support proton transfer during catalysis. Using pulse radiolysis and observing the UV absorbance of superoxide, we have determined rate constants for the catalytic dismutation of superoxide. In addition, the rates of formation and dissociation of the product-inhibited complex of these mutants were determined by direct observation of the characteristic visible absorption of the oxidized and inhibited states. Catalysis by W161A and W161F MnSOD was associated with a decrease of at least 100-fold in the catalytic rate of reduction of superoxide, which then promotes a competing pathway leading to product inhibition. The structural changes caused by the mutations at position 161 led to small changes, at most a 6-fold decrease, in the rate constant for formation of the inhibited complex. Solvent hydrogen isotope effects support a mechanism in which formation of this complex, presumably the peroxide dianion bound to the manganese, involves no rate-contributing proton transfer; however, the dissociation of the complex requires proton transfer to generate HO(2)(-) or H2O2.