Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species.

The contents of extracellular superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase were determined in tissues from nine mammalian species. The pattern of CuZn superoxide dismutase distribution was similar in all species, with high activity in metabolically active organs such as liver and kidney and low activity in, for example, skeletal muscle. Mn superoxide dismutase activity was high in organs with high respiration, such as liver, kidney, and myocardium. Overall the Mn superoxide dismutase activity in organs was almost as high as the CuZn superoxide dismutase activity. The content of extracellular superoxide dismutase was, almost without exception, lower than the content of the other isoenzymes. The pattern of tissue distribution was distinctly different from those of CuZn superoxide dismutase and Mn superoxide dismutase. The tissue distribution of extracellular superoxide dismutase differed among species, but in general there was much in lungs and kidneys and little in skeletal muscle. In man, pig, sheep, cow, rabbit and mouse the overall tissue extracellular superoxide dismutase activities were similar to each other, whereas dog, cat and rat tissues contained distinctly less. There was no general correlation between the tissue extracellular superoxide dismutase activity of any of the various species and the variable plasma activity. The ratio between the plasma and the overall tissue activities was high, for some species over unity, providing further evidence for the notion that one role of extracellular superoxide dismutase is as a plasma protein.     

Regulation of Mn-SOD activity in the mouse heart: glucose effect

Intraperitoneal injection of glucose was found to cause a dose and time dependent suppression of superoxide dismutase activity in mouse heart. Manganese superoxide dismutase was more sensitive to glucose suppression than Cu-Zn superoxide dismutase. While glucose suppressed the Mn form of the enzyme at the concentration of 1.5 mg/kg, it did not have a significant effect on Cu-Zn superoxide dismutase activity at this concentration. The maximum suppression for both forms of superoxide dismutase activity occurred at 4.5 mg/kg. Glucose also suppressed manganese superoxide dismutase activity in mouse heart for a longer period of time compared to Cu-Zn superoxide dismutase. Glucose suppression also occurred in mouse brain. The glucose suppression effect on manganese superoxide dismutase activity in the heart was partially alleviated by X-irradiation.     

Regulation of the synthesis of superoxide dismutases in rat lungs during oxidant and hyperthermic stresses

Heat shock proteins are induced at normal temperatures by oxidants and during reoxygenation following hypoxia. We now report cyanide-resistant O2 consumption increased 30-50% in rat lungs exposed to heat shock or reoxygenation following hypoxia. The synthesis of Cu,Zn superoxide dismutase, but not Mn superoxide dismutase, was increased in rat lung slices by in vivo hyperthermia (39 degrees C), by in vitro heat shock (41 degrees C), and during incubation of lung slices with the Cu chelator diethyldithiocarbamate, which decreased the activity of Cu,Zn superoxide dismutase. The heat shock-induced increase in Cu,Zn superoxide dismutase developed 2 h later than the induction of heat shock proteins and was not blocked by actinomycin D. The rates of synthesis of both superoxide dismutases were decreased 50% by hypoxia and failed to increase during reoxygenation. During hypoxia the activity of Cu,Zn superoxide dismutase decreased about 50%, but the activity of Mn superoxide dismutase remained unchanged. We conclude that hyperthermia increases the synthesis of Cu,Zn superoxide dismutase, the synthesis of Cu,Zn superoxide dismutase and Mn superoxide dismutase are not coordinately regulated by hyperthermia or by the oxidant stress produced by lowering the activity of Cu,Zn superoxide dismutase, and the synthesis of heat shock proteins and Cu,Zn superoxide dismutase are regulated at different levels of gene expression.     

Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes

Leishmania tropica, Trypanosoma brucei, Trypanosoma cruzi, and Crithidia fasciculata have superoxide dismutases which are insensitive to cyanide and sensitive to peroxide and azide, properties characteristic of iron-containing superoxide dismutase. Studies on the superoxide dismutase of C. fasciculata have revealed that: 1) the enzyme is located in the cytosol; 2) isozymes exist; 3) the major superoxide dismutase isozyme (superoxide dismutase 2) has Mr approximately equal to 43,000 and consists of two equal-sized subunits, each of which contains 1.4 atoms of iron. Comparisons of the amino acid content of this crithidial superoxide dismutase with those of superoxide dismutases from other sources suggests that the crithidial enzyme is closely related to bacterial Fe-containing superoxide dismutases, and only distantly related to human Mn- and Cu,Zn-containing superoxide dismutases and to Euglena Fe-containing superoxide dismutase. Attempts are now underway to develop specific inhibitors of the trypanosomatid superoxide dismutase which may be of use in the treatment of leishmaniasis or trypanosomiasis.     

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.     

Superoxide dismutase-rich bacteria. Paradoxical increase in oxidant toxicity

Superoxide dismutase is considered important in protection of aerobes against oxidant damage, and increased tolerance to oxidant stress is associated with induction of this enzyme. However, the importance of superoxide dismutase in this tolerance is not clear because conditions which promote the synthesis of superoxide dismutase likewise affect other antioxidant enzymes and substances. To clarify the role of superoxide dismutase per se in organismal defense against oxidant-generating drugs, we employed Escherichia coli transformed with multiple copies of the gene for bacterial iron superoxide dismutase. These bacteria have greater than ten times the superoxide dismutase activity of wild-type E. coli but, importantly, are normal in other oxidant defense parameters including catalase, peroxidases, glutathione, and glutathione reductase. High superoxide dismutase and control bacteria were exposed to the O2- -generating drug paraquat and to elevated pO2. We find; high superoxide dismutase E. coli are more readily killed by paraquat under aerobic, but not anaerobic, conditions. During exposure to paraquat, high superoxide dismutase E. coli accumulate more H2O2. Coincidentally, the reduced glutathione content of high superoxide dismutase E. coli declines more than in control E. coli. E. coli with high superoxide dismutase activity are also more readily killed by hyperoxia. Interestingly, the susceptibility of the parental and high superoxide dismutase E. coli to killing by exogenous H2O2 is not significantly different. Thus, under these experimental conditions, greatly enhanced superoxide dismutase activity accelerates H2O2 formation. The increased H2O2 probably accounts for the exaggerated sensitivity of high superoxide dismutase bacteria to oxidant-generating drugs. These results support the concept that the product of superoxide dismutase, H2O2, is at least as hazardous as the substrate, O2-. We conclude that effective organismal defense against reactive oxygen species may require balanced increments in antioxidant enzymes and cannot necessarily be improved by increases in the activity of single enzymes.     

A rapid whole-cell assay for superoxide dismutase

A procedure is described for the intact-cell assay of superoxide dismutase(s). The technique involves the use of toluene which renders the cells permeable to the necessary components of a photochemical assay for superoxide dismutase. Whole-cell superoxide dismutase activities from a number of procaryotic and eucaryotic microorganisms compare with cell-free activities and with activities reported in the literature. Using this procedure, changing levels of superoxide dismutase are readily monitored under conditions known to modulate superoxide dismutase activity assayed in vitro. In whole cells of Escherichia coli, exogenous methyl viologen causes a marked increase in superoxide dismutase activity, whereas in the cyanobacterium, Microcystis aeruginosa, such treatment leads to a marked, light-dependent loss of whole-cell superoxide dismutase activity.     

Expression of transfected human CuZn superoxide dismutase gene in mouse L cells and NS20Y neuroblastoma cells induces enhancement of glutathione peroxidase activity

The human CuZn superoxide dismutase (superoxide dismutase 1) a key enzyme in the metabolism of oxygen free-radicals, is encoded by a gene located on chromosome 21 in the region 21 q 22.1 known to be involved in Down's syndrome. A gene dosage effect for this enzyme has been reported in trisomy 21. To assess the biological consequences of superoxide dismutase 1 overproduction within cells, the human superoxide dismutase 1 gene and a human superoxide dismutase 1 cDNA were introduced into mouse L cells and NS20Y neuroblastoma cells. Both cell types expressed elevated levels (up to 3-fold) of enzymatically active human superoxide dismutase 1. These human superoxide dismutase 1 overproducers, especially neuronal cell lines, showed an increased activity in the selenodependent glutathione peroxidase. These data are consistent with the possibility that gene dosage of superoxide dismutase 1 contributes to oxygen metabolism modifications previously described in Down's syndrome.     

In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate.

Superoxide dismutase was assayed by a method which takes advantage of the inhibitory action of superoxide dismutase (or tissues which contain superoxide dismutase) on the rate of autooxidation of 6-hydroxydopamine. Incubation of pure superoxide dismutase of homogenates of brain or liver with 10(-3) M diethyldithiocarbamate for 1.5 hours resulted in total loss of superoxide dismutase activity. Inhibition of superoxide dismutase was not reversed by dialysis, but after dialysis, enzymatic activity was restored with CuSO4. When 1.5 g of diethyldithiocarbamate/kg were injected into mice, the superoxide dismutase activity at 3 hours was decreased by 86%, 71%, and 48%, respectively, in whole blood, liver, and brain. A dose of 0.5 g of diethyldithiocarbamate/kg lowered the superoxide dismutase activity by 42% in liver at 3 hours. A study of the time course for inhibiton of superoxide dismutase in liver after 1.5 g of diethyldithiocarbamate/kg, showed a maximum decrease (81%) within 1 hour, with a slow return to 64% of normal by 24 hours. Inhibition of superoxide dismutase in vivo and in vitro was confirmed with other assay systems based on the autooxidation of pyrogallol or epinephrine or on reduction of cytochrome c or intro blue tetrazolium. Treatment of animals with diethyldithiocarbamate may provide a useful experimental model to study the role of superoxide dismutase in various tissues.     

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.     

Endocytosis of superoxide dismutase is required in order for the enzyme to protect hepatocytes from the cytotoxicity of hydrogen peroxide

Inhibitors of endocytosis have been used to show that internalization of superoxide dismutase is required for the enzyme to protect hepatocytes from the cytotoxicity of hydrogen peroxide. As shown previously (Starke, P. E., and Farber, J. L. (1985) J. Biol. Chem. 260, 10099-10104), superoxide dismutase prevented the killing of cultured hepatocytes by H2O2 generated in the medium by glucose oxidase. Five inhibitors of endocytosis, methylamine, monensin, benzyl alcohol, cytochalasin B, and oligomycin, each abolished the protective effect of superoxide dismutase. Cell-associated superoxide dismutase activity was increased 4-fold in hepatocytes after exposure to superoxide dismutase for 1 h. Each of the inhibitors abolished this increase in the cell-associated superoxide dismutase activity. The uptake of horseradish peroxidase, a measure of fluid phase endocytosis, differed from that of superoxide dismutase in its lower rate, reduced sensitivity to methylamine, and its insensitivity to cytochalasin B. The results of the present study demonstrate that endocytosis of superoxide dismutase is required to protect hepatocytes from the cytotoxicity of hydrogen peroxide. This conclusion may account for some of the conflicting results in the literature with respect to the protective action of superoxide dismutase.     

A highly sensitive method for determining both Mn- and Cu-Zn superoxide dismutase activities in tissues and blood cells

A highly sensitive method for determining the superoxide dismutase (EC 1.15.1.1) in various tissues and blood cells is described. This method involves inhibition of a cypridina luciferin analog that is chemiluminescence dependent upon O2- generated by hypoxanthine-xanthine oxidase. Manganeous superoxide dismutase, which is sensitive to sodium dodecyl sulfate, was determined and calculated by subtraction of superoxide dismutase activity in tissue extract treated with this detergent (Cu-Zn superoxide dismutase) from that in untreated tissue extract (total superoxide dismutase). Both Mn- and Cu-Zn superoxide dismutase activities were expressed as equivalent nanograms of bovine erythrocyte superoxide dismutase per milliliter. Sensitivity limits of the chemiluminescence methods with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one as cypridina luciferin analogs were 1 ng and 2-3 ng of superoxide dismutase/ml, respectively.     

Superoxide dismutase isoenzymes in tissues and plasma from New Zealand black mice, nude mice and normal BALB/c mice

In various types of autoimmune disease, an increased frequency of spontaneous chromosome breaks has been reported. Plasma from such patients induces chromosome breaks in normal cells. Exposure of plasma to superoxide radicals increases the breakage activity, and addition of superoxide dismutase protects against it. The New Zealand black mouse is an animal model of autoimmune disease which displays the breakage phenomenon. To test for the possibility that the breakage is related to deficient protection against superoxide radicals, the activities of superoxide dismutase isoenzymes were determined in tissues and blood from New Zealand black mice and compared with the activities of normal BALB/c mice. No differences between the strains were revealed in tissue EC-superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase activity. The erythrocyte superoxide dismutase activities were also equal. The plasma EC-superoxide dismutase activity was 35% lower in the New Zealand black mice than in the BALB/c mice. Between euthymic BALB/c mice and nude mice, previously reported to be deficient in tissue superoxide dismutase activity, no difference could be demonstrated.     

Alteration of endogenous glutathione peroxidase, manganese superoxide dismutase, and glutathione transferase activity in cells transfected with a copper-zinc superoxide dismutase expression vector. Explanation for variations in paraquat resistance

Transfection of a human pSV2 (copper-zinc) superoxide dismutase expression vector into murine fibroblasts resulted in stable clones producing increased amounts of copper-zinc superoxide dismutase. A marked increase in endogenous glutathione peroxidase activity (up to 285%) and a smaller increase in glutathione transferase activity (up to 16%) also occurred. Manganese superoxide dismutase activity was decreased in all clones, whereas catalase and NADPH reductase activities were not affected. Alterations in glutathione peroxidase and manganese superoxide dismutase activities correlated with increases in copper-zinc superoxide dismutase activity. Whereas all clones were resistant to paraquat, a direct correlation between copper-zinc superoxide dismutase activity and resistance to paraquat did not exist. In agreement with previous reports clones expressing the highest copper-zinc superoxide dismutase activity did not display the highest resistance to paraquat. However, there was a direct correlation between the increase in glutathione peroxidase activity and paraquat resistance (p less than 0.002).     

Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage.

1. Activities of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H2O2 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-1H-1,2,4-triazole (3-aminotriazole) or NaN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H2O2. 4. In rabbit having early cataract (vacuolar stage) induced by feeding-3-aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-Fold increase in H2O2 of aqueous humor and vitreous humor. We conclude that catalase of eye affords protection to the lens from H2O2 and it also protects superoxide dismutase of lens from inactivation by H2O2. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2.-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the hydroxyl radical, under pathological conditions when H2O2 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.     

Demonstration of Cu-Zn superoxide dismutase in rat liver peroxisomes. Biochemical and immunochemical evidence.

In this study, by using highly purified rat liver peroxisomes, we provide evidence from analytical cell fractionation, Western blot, and immunocytochemical analysis that Cu-Zn superoxide dismutase is present in animal peroxisomes. Treatment with ciprofibrate, a peroxisome proliferator, increased the peroxisomal superoxide dismutase activity by 3-fold with no effect on mitochondrial activity but a marked decrease in cytosolic superoxide dismutase activity, further supporting that besides cytosolic and mitochondrial localization, Cu-Zn superoxide dismutase is present in peroxisomes also. Demonstration of superoxide dismutase in peroxisomes suggests a new role for this organelle in pathophysiological conditions, such as ischemia-reperfusion injury.     

Superoxide dismutase activity in early stages of development in normal and dystrophic chickens

Changes in superoxide dismutase activities in early stages of chronological development were investigated in normal and dystrophic chickens. Both cupro-zinc and manganese superoxide dismutase activities were significantly elevated in the dystrophic chickens studied as early as one week after hatching compared to those in the control. In control chickens, both cupro-zinc and manganese superoxide dismutase activities declined as they grew older. In dystrophic chickens, manganese superoxide dismutase activity declined gradually as they grew older as in the control. However, cupro-zinc superoxide dismutase activity increased until four weeks of age. The latter activity was still twice as high as that of the control at four months of age. Increased activities in superoxide dismutases in early stages of the development suggest presence of increased turnover of active oxygen species from the early stage of the disease in this avian muscular dystrophy. And the distinct time course of cupro-zinc superoxide dismutase activity suggests involvement of active oxygen species in pathogenesis of this disorder.     

Engineering and characterization of human manganese superoxide dismutase mutants with high activity and low product inhibition

Human manganese superoxide dismutase is a mitochondrial metalloenzyme that is involved in protecting aerobic organisms against superoxide toxicity, and has been implicated in slowing tumor growth. Unfortunately, this enzyme exhibits strong product inhibition, which limits its potential biomedical applications. Previous efforts to alleviate human manganese superoxide dismutase product inhibition utilized rational protein design and site-directed mutagenesis. These efforts led to variants of human manganese superoxide dismutase at residue 143 with dramatically reduced product inhibition, but also reduced catalytic activity and efficiency. Here, we report the use of a directed evolution approach to engineer two variants of the Q143A human manganese superoxide dismutase mutant enzyme with improved catalytic activity and efficiency. Two separate activity-restoring mutations were found--C140S and N73S--that increase the catalytic efficiency of the parent Q143A human manganese superoxide dismutase enzyme by up to five-fold while maintaining low product inhibition. Interestingly, C140S is a context-dependent mutation, and the C140S-Q143A human manganese superoxide dismutase did not follow Michaelis-Menten kinetics. The re-engineered human manganese superoxide dismutase mutants should be useful for biomedical applications, and our kinetic and structural studies also provide new insights into the structure-function relationships of human manganese superoxide dismutase.     

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.     

Antifibrotic effects of aldehyde dextran modified superoxide dismutase in experimental silicosis]

The present paper dwells on biomedical study of aldehyde dextran modified superoxide dismutase. Pharmacokinetic data demonstrated that modification of superoxide dismutase increased its half-time. A rat model of experimental silicosis showed that aldehyde dextran modified superoxide dismutase inhibited evolving fibrosis in the lungs. The same dose of native enzyme produced no therapeutic effect. Thus, superoxide dismutase can be considered as a potential agent for treatment of fibrosis due to its modification.