Rat superoxide dismutases. Purification, labeling, immunoassay, and tissue concentration

This study determines the validity of utilizing radioimmunoassay of CuZn and Mn superoxide dismutase in the rat for defining mechanism of control over mammalian tissue superoxide dismutase concentrations. To accomplish this, rat Mn and CuZn superoxide dismutase were purified. The CuZn superoxide dismutase dimer had a specific activity of 3600 units/mg of protein and a subunit Mr of 17,000. The Mn superoxide dismutase tetramer had a specific activity of 3700 units/mg of protein and a subunit Mr of 22,000. Both enzymes provided a single discrete protein band on disc gel electrophoresis. The purified enzymes were utilized to develop sensitive (less than 2.5 ng/ml Mn superoxide dismutase and less than 3.12 ng/ml CuZn superoxide dismutase) reproducible immunoassays the specificity of which was confirmed by tissue homogenate dilution and column chromatography. Immunoassay of these enzymes in rat tissues permitted clarification of existing data based on activity assays and demonstrated a trend for higher Mn superoxide dismutase concentrations in tissues of high mitochondrial content (with relative tissue concentrations comparable to man) and low superoxide dismutase concentrations in islets (providing an explanation for their sensitivity to free radical damage). This represents the first report of a radioimmunoassay for rat Mn superoxide dismutase, and the second report of successful purification of rat Mn superoxide dismutase (with higher specific activity and apparent purity and stability). The data support the proposition that these radioimmunoassays in rats will provide a useful system for investigation of mechanisms of control over tissue superoxide dismutase concentrations in mammalian tissues.     

Expression of antioxidant enzymes in rat kidney during ischemia-reperfusion injury

The effect of ischemia-reperfusion on activity, protein and m-RNA levels of catalase, copper-zinc and manganese containing superoxide dismutases and glutathione peroxidase, the enzymes that are involved in free radical detoxification was studied in rat kidney. Ischemia alone did not alter either the activities or protein levels of superoxide dismutase and glutathione peroxidase. However, catalase activity was found to be inhibited to 82% of control. The inhibition of catalase was due to the inactivation of the enzyme as there was no significant change in enzyme protein level. Reperfusion following ischemia, however, led to a significant decrease in both the activities as well as the protein levels of all the antioxidant enzymes. The observed overall decrease in total superoxide dismutase activity was the net effect of a decrease in copper-zinc superoxide dismutase while manganese superoxide dismutase activity was found to be increased following reperfusion. This observed increased manganese superoxide dismutase activity was the result of its increased protein level. The mRNA levels for catalase, superoxide dismutases, and glutathione peroxidase were observed to be increased (100–145% of controls) following ischemia; reperfusion of ischemic kidneys, however, resulted in a significant decrease in the levels of mRNAs coding for all the enzymes except manganese superoxide dismutase which remained high. These results suggest that in tissue, the down regulation of the antioxidant enzyme system could be responsible for the pathophysiology of ischemia-reperfusion injury.     

Properties of spermatozoal superoxide dismutase and lack of involvement of superoxides in metal-ion-catalysed lipid-peroxidation and reactions in semen.

1. The distribution and properties of superoxide dismutase were examined in mammalian semen, and the enzyme was used to investigate the role of superoxides in metal-ion-catalysed lipid-peroxidation reactions in spermatozoa. 2. Superoxide dismutase activity was detected in seminal plasma and spermatozoa from all species studied, exceptionally high activity being found in donkey semen. The enzyme is easily solubilized from spermatozoa, as 85-90% of the total activity is released by cold shock, a relatively mild form of cellular damage. 3. Purification and characterization of the enzyme from supernatant fractions prepared from cold-shocked boar spermatozoa showed it to be cyanide-sensitive, to have a mol.wt. of 31 000, a pI of 5.9 and to contain 1.85 g-atoms of copper and 1.91 g-atoms of zinc per mol of protein. However, extensive sonication of spermatozoa released a small amount of a cyanide-insensitive enzyme, presumably a mangano superoxide dismutase, from the mitochondrial matrix. 4. The presence of superoxide dismutase in spermatozoa, either intracellularly or extracellularly, did not inhibit ascorbate/Fe2+-catalysed lipid-peroxidation reactions, suggesting that superoxides are not essential intermediates in this system.     

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.     

Dietary orotic acid affects antioxidant enzyme mRNA levels and oxidative damage to lipids and proteins in rat liver

We investigated the effects of the dietary addition of orotic acid on liver antioxidant enzymes, mRNA levels of these enzymes, and peroxidative products by comparing casein with soy protein as the source of dietary protein. Rats fed the casein diet accumulated more liver lipids than those fed the soy protein diet when orotic acid was added. The addition of orotic acid lowered both the activity of liver Cu, Zn-superoxide dismutase and the level of Cu, Zn-superoxide dismutase mRNA. The addition of orotic acid led to a significant increase in the contents of conjugated dienes and protein carbonyls in the liver. In addition, dietary soy protein protected the increase in the levels of lipids and proteins peroxide induced by orotic acid. The addition of orotic acid to the casein diet increased the activities of both serum ornithine carbamoyltransferase and alanine aminotransferase. Thus, liver damage might result from the increased superoxide anion due to the decrease in the activity of hepatic superoxide dismutase, as well as increase in the production of hepatic peroxidative products in rats fed the casein diet with orotic acid.     

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.     

Immunocytochemical evidence for a peroxisomal localization of manganese superoxide dismutase in leaf protoplasts from a higher plant

The controversial question of the intracellular location of manganese-containing superoxide dismutase in higher plants was examined under a new experimental approach by applying the more rigorous and specific methods of immunocytochemistry to protoplasts isolated fromPisum sativum L. leaves. Manganese superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from 15 kg of leaves ofPisum sativum L. Rabbits were immunized with the mangano enzyme and the antibody specific for pea manganese superoxide dismutase was purified and found not to contain antigenic sites in common with (i) human manganese superoxide dismutase, (ii) iron superoxide dismutase from eitherEscherichia coli or higher plants, or (iii) plant or animal cuprozinc-superoxide dismutase.Pisum sativum L. manganese superoxide dismutase only appears to have antigenic determinants similar to other manganese superoxide dismutases from higher land plants. The antibody to pea Mn-superoxide dismutase was used to locate the enzyme in protoplasts isolated from young pea leaves by indirect immunofluorescence, and by electron microscopy using the unlabelled antibody peroxidase-antiperoxidase method. Results from immunofluorescence showed that chloroplasts were devoid of specific fluorescence which appeared scattered over the cytosolic spaces among chloroplasts, and demonstrate the absence of manganese superoxide dismutase inside chloroplasts. The metalloenzyme was found to be localized only in peroxisomes, whereas mitochondria, the traditionally accepted site for this enzyme in many eukaryotic organisms, did not show any specific staining. The possible subcellular roles of manganese superoxide dismutase inPisum sativum L. leaves are discussed in the light of its peroxisomal location.     

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.     

Partial purification of superoxide dismutase and peroxidase from ber (Zizyphus mauritiana Lamk.) fruit using anion exchange chromatography

Partial purification of the two enzymes i.e. superoxide dismutase (SOD) and peroxidase (POX) from ber pulp has been obtained by passing the ammonium sulphate fraction through a diethyl amino ethyl-cellulose (DEAE-Cellulose) column. The fractions showing SOD and POX activity were pooled separately and passed through a Sephadex G100 column for further purification. SOD was purified 12.2 fold with 12.6% yield while POX was purified 15.6 fold with 19.3% yield. Approximate molecular mass for SOD and POX, as judged by gel filtration method was 35.6 and 81.5 kDa, respectively.Key words: Ber fruit, superoxide dismutase, peroxidase, DEAE-cellulose chromatography, fold purification, yield, Zizyphus mauritiana Lamk     

小胸鳖甲胞外铜锌超氧化物歧化酶重组蛋白的原核表达及多克隆抗体制备

超氧化物歧化酶(SOD)是清除生物体内超氧阴离子自由基的主要抗氧化酶家族。基于原核表达系统,成功表达了拟步甲科Tenebrionidae小胸鳖甲Micordera punctipennis胞外铜锌SOD的重组蛋白(本文定义为Trx-His-MpecCu/Zn-SOD)。经Ni 2+亲和层析法纯化重组蛋白后,研究了重组蛋白的部分酶学性质。通过足垫加皮下注射法3次免疫小鼠后,分别用ELISA和Western blot的方法检测抗体效价和抗体特异性。结果表明,重组蛋白主要以包涵体形式存在,纯化后的重组蛋白浓度为1.33 mg·mL^-1,酶活力为27.52 U·mg^-1。Trx-His-MpecCu/Zn-SOD在25~45℃具有比较稳定的酶活性,在35℃最高,同时表现出比较广泛的酸碱耐受性(pH3~12),最适pH为9.0,表明重组蛋白的酶活性相对比较稳定。蛋白免疫法制备的鼠抗MpecCu/Zn-SOD多克隆抗体滴度高于1∶819 200。Western blot结果显示,该抗体能免疫结合重组蛋白Trx-His-MpecCu/Zn-SOD和小胸鳖甲体内天然MpecCu/Zn-SOD,但不能与黄粉虫Tenebrio molitor的总蛋白结合,说明制备的抗体效价较高且特异性较好。本研究结果为小胸鳖甲ecCu/Zn-SOD功能的深入研究奠定了基础。     

Differential Effect of Denervation on Free-Radical Scavenging Enzymes in Slow and Fast Muscle of Rat

To determine the effect of denervation on the free-radical scavenging systems in relation to the mitochondrial oxidative metabolism in the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles, the sciatic nerve of the rat was crushed in the midthigh region and the muscle tissue levels of five enzymes were studied 2 and 5 weeks following crush. Recently developed radioimmunoassays were utilized for the selective measurement of cuprozinc (cytosolic) and mangano (mitochondrial) superoxide dismutases. Total tissue content of cuprozinc superoxide dismutase showed a mild decrease after denervation in slow but not in fast muscle. Manganosuperoxide dismutase and fumarase decreased markedly at 2 weeks and returned toward control levels by 5 weeks, the changes appearing to be greater in slow than in fast muscle. At 2 weeks, cytochrome c oxidase decreased significantly in slow, but not in fast muscle. GSH-peroxidase at baseline was 10-fold higher in slow than in fast muscle, markedly decreased at 2 weeks in slow muscle, and returned toward control levels at 5 weeks, whereas the total enzyme activity in fast muscle did not change through 5 weeks. These data represent the first systematic report of free radical scavenging systems in slow and fast muscles in response to denervation. Selective modification of cuprozinc and manganosuperoxide dismutases and differential regulation of GSH-peroxidase was demonstrated in slow and fast muscle.     

Effect of heavy metals on wheat seedlings; activation of antioxidant enzymes

Accumulation of heavy metals in wheat grain exposed to multicomponent pollutants (industrial waste-water) was studied. The absolute content of metals (Zn, Cd, Cu, Cr, Ni, Pb, and Mn) was found to be determined by the extent of purification of wastewater. An increase in the degree of grain contamination with heavy metals was accompanied by activation of antioxidant enzymes (superoxide dismutase, EC 1.15.1; catalase, EC 1.11.1.6; and peroxidase, EC 1.11.1.7) in leaves and activation of superoxide dismutase and peroxidase in roots. The ratio of activity of membrane enzymes to activity of cytosol enzymes was demonstrated to be high. It was concluded that the membrane-tropic effect of multicomponent contaminants was due to accumulation of heavy metals capable of inducing the antioxidant protection in the next generation of wheat seedlings.     

Effect of Heavy Metals on Wheat Seedlings: Activation of Antioxidant Enzymes

Accumulation of heavy metals in wheat grain exposed to multicomponent pollutants (industrial wastewater) was studied. The absolute content of metals (Zn, Cd, Cu, Cr, Ni, Co, Pb, and Mn) was found to be determined by the extent of purification of wastewater. An increase in the degree of grain contamination with heavy metals was accompanied by activation of antioxidant enzymes (superoxide dismutase, EC 1.15.1.1; catalase, EC 1.11.1.6; and peroxidase, EC 1.11.1.7) in leaves and activation of superoxide dismutase and peroxidase in roots. The ratio of activity of membrane enzymes to activity of cytosol enzymes was demonstrated to be high. It was concluded that the membranotropic effect of multicomponent contaminants was due to accumulation of heavy metals capable of inducing the antioxidant protection in the next generation of wheat seedlings.     

Purification of soluble enzymes from erythrocyte hemolysates by three phase partitioning

1. The three phase partitioning method of protein fractionation was successfully applied to human erythrocyte hemolysates for the removal of hemoglobin and the concentration of soluble enzymes. 2. Human carbonic anhydrase I and II, catalase and superoxide dismutase were recovered free of hemoglobin and in good yield in the initial partitioning step, with a 60- to 80-fold enrichment of enzyme activities. 3. After further purification, carbonic anhydrases I and II were obtained at overall yields of 84 and 29%, respectively, crystallized catalase at 38% and superoxide dismutase at 52%.     

The amino acid sequence of mangano superoxide dismutase from Escherichia coli B.

The complete amino acid sequence of the mangano superoxide dismutase from Escherichia coli B has been deduced through characterization of peptides from cyanogen bromide, bromonitrophenylsulfenyl skatole, citraconylated tryptic, and succinylated tryptic digests of the intact polypeptide chain and through subfragmentation of selected peptides with chymotrypsin, thermolysin, trypsin, and Staphylococcus aureus V8 extracellular protease. No significant homology is detected on comparison with the sequence of the copper- and zinc-containing superoxide dismutase from bovine erythrocytes, indicating that the manganese-iron and the copper-zinc classes of dismutases arose from independent evolutionary ancestors, a proposal previously based solely on enzymological and NH2-terminal sequence data. The amino acid sequence listed below corresponds to a molecular weight of 22,900 and appears to be identical in each subunit polypeptide of the native enzyme dimer. formula: (see text).     

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.     

Superoxide dismutase in wounded etiolated pea seedlings

The activities of three related enzymes, superoxide dismutase, peroxidase and catalase, were followed in decapitated (wounded) etiolated pea stem tissue. Of the three enzyme activities, only that of superoxide dismutase showed a significant loss and recovery pattern within 1 hr. The change in enzymatic activity appears to result from protein loss and recovery.     

The effect of hyperbaric oxygenation on the antioxidant status of Xenopus laevis after its preliminary adaptation to oxygen]

We studied the level of lipid peroxidation and the activity of antioxidant enzymes (superoxide dismutase and catalase) in various tissues of adult Xenopus laevis after an initial exposure to hyperbaric oxygenation at the developmental stage 38. We have found that irrespective to the mode of treatment, the level of lipid peroxidation and activity of antioxidant enzymes in the brain, lungs, and blood of these animals were higher as compared to control animals. We demonstrate that, after the exposure of adult animals to hyperoxia, if they were earlier subjected to hyperbaric oxygenation (0.2 MPa) at stage 38, there was no intensification of lipid peroxidation or changes in the activity of superoxide dismutase and catalase. In adult animals initially subjected to hyperbaric oxygenation at the same stage of development but at the pressure--0.7 MPa, the second exposure to hyperoxia led to a drastic intensification of lipid peroxidation in the brain; in some animals, an increased level of lipid peroxidation products in the lungs was observed.     

Melatonin protects against delta-aminolevulinic acid-induced oxidative damage in male Syrian hamster Harderian glands

Effects of the prooxidant delta-aminolevulinic acid (ALA) and the antioxidant melatonin (MEL) were investigated in the male Syrian hamster Harderian gland (HG). Rodent Harderian glands are highly porphyrogenic organs, which may be used as model systems for studying damage by delta-aminolevulinic acid and its metabolites, as occurring in porphyrias. Chronic administration of delta-aminolevulinic acid (2 weeks) markedly decreased activities of the porphyrogenic enzymes delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) and of the antioxidant enzymes superoxide dismutase (SOD), glutathione reductase (GR) and catalase (CAT), whereas porphobilinogen deaminase (PBG-D) remained unaffected. This treatment led to increased lipid peroxidation (LPO) and oxidatively modified protein (protein carbonyl) as well as to morphologically apparent tissue damage. Melatonin also caused decreases in delta-aminolevulinate synthase, delta-aminolevulinate dehydratase, superoxide dismutase, glutathione reductase and catalase. Despite lower activities of antioxidant enzymes, lipid peroxidation and protein carbonyl were markedly diminished. The combination of delta-aminolevulinic acid and melatonin led to approximately normal levels of delta-aminolevulinate dehydratase, glutathione reductase, catalase and protein carbonyl, and to rises in superoxide dismutase and porphobilinogen deaminase activities; lipid peroxidation remained even lower than in controls and the appearance of the tissue revealed a protective influence of melatonin. These results suggest that melatonin may have profound effects on the oxidant status of the Harderian gland.     

Biosynthesis of oxygen-detoxifying enzymes in Bdellovibrio stolpii.

Axenically grown Bdellovibrio stolpii (i.e., grown independently of the host) was examined for superoxide dismutase, catalase, and peroxidase activities. Kinetics of enzyme synthesis were determined for aerobically grown cultures and for cultures exposed to 100% oxygen. Enzymatic activities varied with the age of the culture. Normally grown cultures exhibited maximum activity during the first 10 h of growth and again as the stationary phase was approached, beginning at about 48 h. Polyacrylamide gel electropherograms of cell-free extracts revealed that B. stolpii contained one major band (1) and two minor bands (II, III) of superoxide dismutase activity. Each of these enzymes was inactivated by H2O2, indicating that they were iron-containing enzymes. Manganese-containing superoxide dismutase was not detected in B. stolpii. Increased oxygenation did not appreciably stimulate enzyme synthesis, for only superoxide dismutase was induced, reaching maximum activity at 10 h and then rapidly falling to normal levels. Superoxide dismutase appears to be the main enzymatic defense against oxygen toxicity in B. stolpii. Induction of superoxide dismutase with 100% oxygen was manifested as an increase in the intensities of the two minor bands of activity, suggesting that isozyme I is constitutive, whereas isozymes II and III are inducible. The induction of isozymes II and III by 100% oxygen was prevented by an inhibitor of protein biosynthesis, chloramphenicol.