Protection of Methanosarcina barkeri against oxidative stress: identification and characterization of an iron superoxide dismutase

Methanosarcina barkeri is a methanogenic archaeon that can only grow under strictly anoxic conditions but which can survive oxidative stress. We have recently reported that the organism contains a monofunctional catalase. We describe here that it also possesses an active iron superoxide dismutase. The enzyme was purified in three steps over 130-fold in a 14% yield to a specific activity of 1500 U/mg. SDS-PAGE revealed the presence of only one band, at an apparent molecular mass of 25 kDa. The primary structure determined from the cloned and sequenced gene revealed similarity to iron- and manganese superoxide dismutases. The highest similarity was to the iron superoxide dismutase from Methanobacterium thermoautotrophicum. The enzyme from M. barkeri was found to contain, per mol, 1 mol iron, but no manganese in agreement with the general observation that anaerobically growing organisms only contain iron superoxide dismutase. The enzyme was not inhibited by cyanide (10 mM), which is a property shared by all iron- and manganese superoxide dismutases. The presence of superoxide dismutase in M. barkeri is noteworthy since a gene encoding superoxide dismutase (sod) has not been found in Archaeoglobus fulgidus, a sulfate-reducing archaeon most closely related to the Methanosarcinaceae.     

Differential temperature sensitivity of pea superoxide dismutases

The activity of pea (Pisum sativum L.) Cu/Zn and Mn superoxide dismutase isoforms was evaluated across a range of temperatures from 10 to 45°C. Maximal activity of the Cu/Zn and Mn superoxide dismutase isoforms was observed at 10°C. Both cytoplasmic and chloroplast Cu/Zn superoxide dismutases exhibit a reduction in staining intensity with increasing temperatures. Mn superoxide dismutase, however, maintained a relatively constant staining intensity across the range of temperatures evaluated. An unrelated enzyme used as a control, malate dehydrogenase, exhibited the expected increase in staining activity with increasing temperatures. These results describe a unique response of a protection enzyme to temperature.     

Corynebacterium glutamicum superoxide dismutase is a manganese-strict non-cambialistic enzyme in vitro

Superoxide dismutase (SOD) of Corynebacterium glutamicum was purified and characterized. The enzyme had a native molecular weight of about 80kDa, whereas a monomer with molecular weight of 24kDa was found on SDS-PAGE suggesting it to be homotetramer. The native SOD activity stained gel revealed a unique cytosolic enzyme. Supplementing growth media with manganese increased the specific activity significantly, while adding iron did not result in significant difference. No growth perturbation was observed with the supplemented media. In vitro metal removal and replacement studies revealed conservation of about 85% of the specific activity by substitution with manganese, while substitution with copper, iron, nickel or zinc did not restore any significant specific activity. Manganese was identified by atomic absorption spectrometer, while no signals corresponding to fixing other metallic elements were detected. Thus, C. glutamicum SOD could be considered a strict (non-cambialistic) manganese superoxide dismutase (MnSOD).     

High-level soluble expression of recombinant human manganese superoxide dismutase in Escherichia coli, and its effects on proliferation of the leukemia cell

Manganese superoxide dismutase (Mn-SOD) is one of the major enzymes responsible for the defense against oxidative damage due to reactive oxygen species (ROS) in the mitochondria. The present study aimed to produce and evaluate the genetically engineered manganese superoxide dismutase protein. A recombinant plasmid containing DNA segment coding Mn-SOD protein was transformed into Escherichia coli (E. coli) Rosetta-gami strain, for expression. After induction with IPTG, an expected molecular mass of 25 kDa was detected by SDS-PAGE. After Ni-NTA affinity chromatography purification, the purity rate came up to 95%. UV spectroscopy data for our preparations indicated that a peak at 275 nm existed in the spectrum. SOD activity assay showed that the activity of the rhMn-SOD was 1890.9 U/mg. The ORAC level of rhMn-SOD was 151492.2 uM Trolox equiv/mg. Furthermore, in vitro bioactivity assay indicated that the rhMn-SOD protein can inhibit the proliferation of the leukemia K562 cells.     

Ozone-induced inactivation of antioxidant enzymes

Ozone is an air pollutant that damages a variety of biomolecules. We investigated ozone-induced inactivation of three major antioxidant enzymes. Cu/Zn superoxide dismutase was inactivated by ozone in a concentration-dependent manner. The concentration of ozone for 50% inactivation was approximately 45 microM when 10 microM Cu/Zn superoxide dismutase was incubated for 30 min in the presence of ozone. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was randomly fragmented. Both ascorbate and glutathione were very effective in protecting Cu/Zn superoxide dismutase from ozone-induced inactivation. The other two enzymes, catalase and glutathione peroxidase, were much more resistant to ozone than Cu/Zn superoxide dismutase. The ozone concentrations for 50% inactivation of 10 microM catalase and glutathione peroxidase were 500 and 240 microM, respectively. SDS-PAGE demonstrated that ozone caused formation of high molecular weight aggregates in catalase and dimerization in glutathione peroxidase. Glutathione protected catalase and glutathione peroxidase from ozone but the effective concentrations were much higher than that for Cu/Zn superoxide dismutase. Ascorbate was almost ineffective. The result suggests that, among the three antioxidant enzymes, Cu/Zn superoxide dismutase is a major target for ozone-induced inactivation and both glutathione and ascorbate are very effective in protecting the enzyme from ozone.     

Analysis of extracellular superoxide dismutase in fibroblasts from patients with systemic sclerosis

Systemic sclerosis (SSc) is a chronic disease of connective tissue characterized by vascular damage, autoantibody production and extensive fibrosis of skin, skeletal muscles, vessels and visceral organs. Fibrosis is a biological process involving inflammatory response and reactive oxygen species (ROS) accumulation leading to fibroblast activation. Extracellular superoxide dismutase (SOD3), a copper and zinc superoxide dismutase, which is expressed in selected tissues, is secreted into the extracellular space and catalyzes the dismutation of superoxide radical to hydrogen peroxide and molecular oxygen. Moreover, SOD3 is associated to inflammatory responses in some experimental models. In this paper we analysed, by RT-PCR and immunofluorescence, SOD3 expression and intracellular localization in dermal fibroblasts from both healthy donors and patients affected by diffuse form of SSc. Moreover, we determined SOD3 enzymatic activity in fibroblast culture medium with the xanthine/xanthine oxidase method. Increased expression of SOD3 mRNA was detected in systemic sclerosis fibroblasts (SScF), as compared to control healthy fibroblasts (HF), and SOD3 immunofluorescence staining displayed a characteristic pattern of secretory proteins in both HF and SScF. Superoxide dismutase assay demonstrated that SOD3 enzymatic activity in SScF culture medium is four times more than in HF culture medium. These data suggest that an alteration in SOD3 expression and activity could be associated to SSc fibrosis.     

Effect of iron on expression of superoxide dismutase by Aeromonas salmonicida and associated resistance to superoxide anion

Abstract Superoxide dismutase activity was detected in Aeromonas salmonicida under iron-replete and iron-limited culture conditions. Under iron-replete conditions an iron superoxide dismutase, molecular mass 50,400 Da, was identified based on inhibition by hydrogen peroxide but not by millimolar concentrations of cyanide. When the available iron in the culture medium was limited by addition of the non-assimilable iron chelator 2,2-dipyridyl, a manganese superoxide dismutase, molecular mass 45,600 Da, was identified, which was resistant to inhibition by either hydrogen peroxide or cyanide. The change in enzyme production would appear to be iron dependent, as addition of FeCl₃ in excess to iron-limited broths resulted in only the iron superoxide dismutase being synthesised. Examination of the location of the superoxide dismutase enzymes revealed that the manganese superoxide dismutase expressed under iron limitation is located in the periplasm, while the iron superoxide dismutase has a cytoplasmic location. The periplasmic manganese superoxide dismutase was able to protect A. salmonicida against extracellular riboflavin-generated superoxide, with A. salmonicida grown under iron-limited conditions exhibiting a 32-fold increase in minimum bactericidal concentration of riboflavin compared to cells cultured under iron-replete conditions. Furthermore, in a time-course study of bactericidal activity of exogenously generated superoxide against A. salmonicida , bacteria grown under iron-replete conditions and expressing cytoplasmic iron superoxide dismutase were rapidly killed, whilst those grown under iron limitation expressing periplasmic manganese superoxide dismutase survived for the duration of the experiment.     

Identification, effective purification and functional characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-640

The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-640 was purified using polyethylene glycol fractionation (PEG) and gel-filtration. The cell free extract was subjected to fractionation by PEG-200, 400 and 1500. The 10% (w/v) PEG-1500 gave dextransucrase with maximum specific activity of 23 with 40 fold purification in a single step. The purified enzyme showed multiple molecular forms on SDS-PAGE, however the same sample showed a single band on non-denaturing native-PAGE. The purified dextransucrase fractions obtained from PEG-1500, confirmed the presence of dextran, when run on SDS-PAGE under non-denaturing gels for in situ activity detection by Periodic Acid Schiff's staining. The activity bands corresponded to the native and active form of the purified dextransucrase of approximately, 180kDa molecular size, that appeared on the denaturing gels stained with Coomassie Brilliant Blue. No bands appeared after staining the activity of dextransucrase on non denaturing SDS-PAGE gels with raffinose, which excluded the presence of fructosyltransferases. Further purification of 10% PEG-1500 purified dextransucrase by gel-filtration gave dextransucrase with specific activity of 35 with 61 fold purification.     

Identification of cysteine-containing peptides in protein digests by high-performance liquid chromatography

Preliminary studies which raised questions as to the efficacy of ammonium sulfate precipitation in separating mangano from cuprozinc superoxide dismutase and the adequacy of cyanide sensitivity in monitoring such separation led to the use of electrophoresis with activity and protein staining to monitor purification. This, together with exploitation of the differing isoelectric points of the two enzymes permitting separate elution from the same DE-52 column, led to a simplified method for purification of these enzymes using the same starting material and purification steps initially. The specific activity was 2900 and 3200 units/mg protein and yields were 18 and 20% for mangano and cuprozine superoxide dismutase, respectively.     

In vitro superoxide activity in the haemolymph of the West Indian leaf cockroach,Blaberus discoidalis

The respiratory burst is an NADPH oxidase-driven reduction of molecular oxygen to superoxide, which can occur in phagocytic cells as part of an antimicrobial defence, and is well documented among the vertebrates. This paper describes a process resembling the respiratory burst, which occurs in the haemolymph and haemocytes of the cockroach, Blaberus discoidalis. The in vitro reduction of nitroblue tetrazolium by superoxide to formazan was measured spectrophotometrically in B. discoidalis haemolymph in response to various immune elicitors. Nitroblue tetrazolium reduction was partly impeded in the presence of superoxide dismutase, a specific antioxidant which converts superoxide to hydrogen peroxide, as well as by chemicals known to inhibit the respiratory burst in vertebrates (trifluoperazine, diphenylene iodonium, and N-ethylmaleimide). This suggests the generation of superoxide anions by haemolymph as part of an immune response. Furthermore, formazan staining of elicitor-treated haemocytes was observed microscopically, with less intense staining in the presence of superoxide dismutase. Finally, respiratory burst inhibitors and superoxide dismutase enhanced the growth of E. coli incubated in whole haemolymph, implying a role for haemolymph-derived superoxide in antibacterial defence.     

Superoxide Dismutase Activity in Needles of Norwegian Spruce Trees (Picea abies L.)

The activity of superoxide dismutase was investigated in needles of spruce trees. To obtain maximum activity, needles were homogenized in the presence of Triton X-100 and polyvinylpyrrolidone. Superoxide dismutase activity was measured in dialyzed extracts with a modified epinephrine assay (HP Misra, I Fridovich [1972] J Biol Chem 247: 3170-3175) at pH 10.2. The extracts contained 70 to 120 units of superoxide dismutase per milligram protein. One unit of superoxide dismutase was completely inhibited in the presence of 20 micromolar NaCN. On native polyacrylamide gels three electromorphs were visualized after staining for activity. All three species were sensitive to CN⁻ and H₂O₂ and were therefore assumed to be Cu/Zn-superoxide dismutases. Superoxide dismutase activity was dependent on the age of the needles and declined by approximately 25% within 3 to 4 years.     

大肠埃希菌Mn-SOD基因的克隆、表达及多克隆抗体制备

目的实现Mn-SOD基因在大肠埃希菌中的高可溶性表达,制备Mn-SOD的多克隆抗体。方法用PCR方法从一株野生型大肠埃希菌（E.coli）基因组中扩增Mn-SOD基因编码区．将它克隆到原核表达载体上进行大量表达和纯化,再用纯化的蛋白对新西兰大白兔进行背部多点注射,40d后取其血清,用Western-blot印迹实验测定抗体效果。结果SDS-PAGE分析表明SOD的表达量约为细菌总蛋白的50％;黄嘌呤氧化酶法测定表达蛋白活性,结果表明每毫克菌体可溶性总蛋白中表达产物酶比活为3921．77U／mg,是对照BL21的276．77倍;并制备了高效价的多克隆抗体。结论该研究成功地构建了大肠埃希菌Mn-SOD基因高效原核表达系统,所表达的Mn-SOD具有良好的免疫原性和免疫反应性。     

Superoxide dismutase micro assay in biological material.

A micro assay for the rapid and convenient determination of superoxide dismutase activity in limited amounts of biological material was devised and successfully employed. The combination of the formazan derivative colour formation induced by reaction of O2theta with nitroblue tetrazolium and a suitable analytical polyacrylamide gel electrophoresis system was used. It was possible to show that the reactivity of soluble superoxide dismutases in polyacrylamide gels was proportional to the enzyme concentrations employed. Bovine erythrocyte Cu, Zn-superoxide dismutase (EC 1.15.1.1) (erythrocuprein) served as a standard throughout. To measure the degree of superoxide dismutase activity, a gel-scanning apparatus was usedThe integrated scanning curve of the unstained portions of the gel proved linearly proportional to the logarithm of the superoxide dismutase activity in the range between 10(-12) and 7 X 10(-11) mol of the bovine enzyme. Although the absolute integral is dependent on the different staining conditions, the slope of the superoxide dismutase calibration curve is highly reproducible. Superoxide dismutase added to crude liver and brain homogenates could be fully detected using this assay. Thus, biological material including nucleic acids, enzymes, lipids etc. do not inhibit this reaction.     

Genetically engineered polymers of human CuZn superoxide dismutase. Biochemistry and serum half-lives

CuZn superoxide dismutase is a highly stable dimer of identical subunits with a combined molecular mass of 32,000 daltons. Two human superoxide dismutase genes have been joined in the same translational reading frame, using spacers of different lengths, to encode single chain proteins consisting of two identical human superoxide dismutase subunits. The first construct encodes two directly linked subunits; the terminal glutamine codon of the first gene was changed to a methionine codon and followed immediately by the second gene. The second construct encodes two subunits linked by a 19-amino-acid human immunoglobulin IgA1 hinge sequence. Both constructs produce high levels of catalytically active superoxide dismutase when expressed in Escherichia coli. The protein containing the IgA1 hinge sequence forms polymers up to 750,000 in molecular weight, which are linked together noncovalently by the hydrophobic bonding of the dimer interface. The polymers are soluble, thermostable, and of near normal specific activity. Site-directed in vitro mutagenesis was used to inactivate one of the two human superoxide dismutase subunits. The resulting human superoxide dismutase polymers have approximately 50% activity, thus confirming that the products of both genes are catalytically active. Large amounts of individual polymeric forms have been purified from recombinant yeast and tested for serum stability in rats. The serum half-life is approximately 7 min for both the two-chain wild type human superoxide dismutase dimer (Mr 32,000) and the single chain molecule consisting of a human superoxide dismutase dimer covalently linked by the immunoglobulin hinge region (Mr 34,000), whereas the higher molecular weight polymers (Mr greater than or equal to 68,000) all have half-lives of approximately 145 min.     

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.     

Intracellular utilization of superoxide anion by indoleamine 2,3-dioxygenase of rabbit enterocytes.

The participation of superoxide anion (O2-) in the intracellular indoleamine 2,3-dioxygenase activity was studied using the dispersed cell suspension of the rabbit small intestine. The dioxygenase activity was assayed by measuring [14C]formate released from DL-[ring-2-14C]tryptophan. The addition of diethyldiethiocarbamate, a superoxide dismutase inhibitor, markedly accelerated the intracellular dioxygenase activity while the superoxide dismutase activity decreased concomitantly. Furthermore, substrates of xanthine oxidase such as inosine, adenosine, and hypoxanthine also increased the dioxygenase activity in the cells, particularly in the presence of methylene blue. This increase was completely abolished by the addition of allopurinol, a specific inhibitor of xanthine oxidase. These results, taken together, indicate that the intracellular accumulation of O2- results in acceleration of the in situ dioxygenase activity, and that indoleamine 2,3-dioxygenase utilizes O2- in the isolated intestinal cells.     

修饰SOD及未修饰SOD的稳定性比较研究

测定了分子修饰ＳＯＤ的酶活力及其在不同温度下的稳定性,与未修饰的ＳＯＤ进行了比较,对在不同温度下测定的结果进行数据处理,经推算得出分子修饰ＳＯＤ在２５℃条件下,保存９５％的酶活力达３．５年,保存９０％的酶活力达７．２年,未修饰ＳＯＤ在２５℃条件下,保存９５％的酶活力为１０３ｄ,保存９０％的酶活力为２１３ｄ。     

Identification of superoxide dismutase in rat liver peroxisomes.

In this paper we have investigated whether or not superoxide dismutase is localized in peroxisomes from rat liver. Using an improved method to prepare peroxisomes from clofibrate induced rat livers, we identified superoxide dismutase activity in peroxisomes. This activity was found to be predominantly of the copper-zinc type. The finding of superoxide dismutase activity in peroxisomes makes sense since peroxisomes also contain superoxide generating enzyme activities such as xanthine oxidase.     

Recombinant human extracellular superoxide dismutase produced in milk of transgenic rabbits

Expression of human extracellular superoxide dismutase (EC-SOD), a glycosylated, tetrameric metalloprotein, was targeted to the lactating mammary gland of transgenic rabbits. Efficient expression of the recombinant whey acidic protein/ec-sod gene was achieved and up to 3 mg ml–1 of the enzyme was secreted into the milk. Rabbit milk-produced recombinant EC-SOD was primarily found in the whey and purified by a two-step chromatographic method. To evaluate the rabbit milk-produced human EC-SOD, comparisons with native and Chinese hamster ovary cell (CHO)-produced EC-SOD were performed. All proteins were tetrameric and N-glycosylated. The behaviour on SDS-PAGE and size-exclusion chromatography indicated that the masses, and thereby the extent of post-translational modification of the proteins was similar. The monosaccharide composition of both recombinant EC-SOD variants was analysed and indicated similarities in the attached N-glycans on the two proteins. Furthermore, the peptide maps of the three EC-SOD variants revealed that all proteins had similar polypeptide backbones