Increased activities of antioxidant enzymes and decreased ATP concentration in cultured myoblasts with the 3243A-->G mutation in mitochondrial DNA

The MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is most commonly caused by the 3243A-->G mutation in mitochondrial DNA, resulting in impaired mitochondrial protein synthesis and decreased activities of the respiratory chain complexes. These defects may cause a reduced capacity for ATP synthesis and an increased rate of production of reactive oxygen species. Myoblasts cultured from controls and patients carrying the 3243A-->G mutation were used to measure ATP, ADP, catalase and superoxide dismutase, which was also measured from blood samples. ATP and ADP concentrations were decreased in myoblasts with the 3243A-->G mutation, but the ATP/ADP ratio remained constant, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, and superoxide dismutase activity was slightly, but not significantly higher in the blood of patients with the mutation than in controls. We conclude that impairment of mitochondrial ATP production in myoblasts carrying the 3243A-->G mutation results in adenylate catabolism, causing a decrease in the total adenylate pool. The increase in superoxide dismutase and catalase activities could be an adaptive response to increased production of reactive oxygen species due to dysfunction of the mitochondrial respiratory chain.     

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.     

Increased activities of antioxidant enzymes and decreased ATP concentration in cultured myoblasts with the 3243A→G mutation in mitochondrial DNA

The MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is most commonly caused by the 3243A→G mutation in mitochondrial DNA, resulting in impaired mitochondrial protein synthesis and decreased activities of the respiratory chain complexes. These defects may cause a reduced capacity for ATP synthesis and an increased rate of production of reactive oxygen species. Myoblasts cultured from controls and patients carrying the 3243A→G mutation were used to measure ATP, ADP, catalase and superoxide dismutase, which was also measured from blood samples. ATP and ADP concentrations were decreased in myoblasts with the 3243A→G mutation, but the ATP/ADP ratio remained constant, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, and superoxide dismutase activity was slightly, but not significantly higher in the blood of patients with the mutation than in controls. We conclude that impairment of mitochondrial ATP production in myoblasts carrying the 3243A→G mutation results in adenylate catabolism, causing a decrease in the total adenylate pool. The increase in superoxide dismutase and catalase activities could be an adaptive response to increased production of reactive oxygen species due to dysfunction of the mitochondrial respiratory chain.     

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.     

Liposome-entrapped superoxide dismutase reduces ischemia/reperfusion ‘oxidative stress’ in gerbil brain

Bilateral common carotid artery occlusion (15 min.) followed by two hours of recirculation reduced mitochondrial superoxide dismutase (SOD) and glutathione reductase (GR) activities, and increased susceptibility of mitochondrial membranes to in vitro lipid peroxidation in brain regions (i.e., cortex, striatum and hippocampus) of Mongolian gerbil. Intraperitoneal bolus injection (2 mg/kg b.w.) of liposome-entrapped CuZn superoxide dismutase (l-SOD) increased the endogenous SOD activity in normal brain tissue and, when given at the end of ischemia, counteracted both the ischemic reduction of endogenous SOD and the increased peroxidation of mitochondrial membranes. 1-SOD treatment was ineffective in reducing brain swelling, suggesting that superoxide radicals are not a main participant in the process of (post)ischemic brain edema formation.     

SOD2 overexpression: enhanced mitochondrial tolerance but absence of effect on UCP activity

We have created P1 artificial chromosome transgenic mice expressing the human mitochondrial superoxide dismutase 2 (SOD2) and thus generated mice with a physiologically controlled augmentation of SOD2 expression leading to increased SOD2 enzyme activities and lowered superoxide levels. In the transgenic mice, effects on mitochondrial function such as enhanced oxidative capacity and greater resistance against inducers of mitochondrial permeability were observed. Superoxide in the mitochondrial matrix has been proposed to activate uncoupling proteins (UCPs), thus providing a feedback mechanism that will lower respiratory chain superoxide production by increasing a proton leak across the inner mitochondrial membrane. However, UCP1 and UCP3 activities and mitochondrial ATP production rates were not altered in isolated mitochondria from SOD2 transgenic mice, despite lowered superoxide levels. Globally, the transgenic mice displayed normal resting metabolic rates, indicating an absence of effect on any UCP activities, and normal oxygen consumption responses after norepinephrine injection. These results strongly suggest that endogenously generated matrix superoxide does not regulate UCP activity and in vivo energy expenditure.     

In vitro modulation of antioxidant enzyme levels in normal hamster kidney and estrogen-induced hamster kidney tumor

Antioxidant enzyme (AE) activities were studied in normal hamster kidney proximal tubules and in estrogen-induced hamster kidney cancer. In vivo, kidney tumor had lower activities of manganese superoxide dismutase (MnSOD), copper, zinc superoxide dismutase, catalase, and glutathione peroxidase than kidney proximal tubules. Differences in AE activities were, in general, maintained in tissue culture, with AE activities remaining low in tumor cells compared to normal cells. Normal proximal tubular cells showed significant induction of MnSOD activity as a function of time in culture of following exposure to diethylstilbestrol, a synthetic estrogen, while MnSOD activity remained low in tumor cells under these conditions. Our results suggest that antioxidant enzymes, particularly MnSOD, are regulated differently in estrogen-induced hamster kidney tumor cells than in normal kidney proximal tubular cells, demonstrating that cancers arising from hormonal influence have similar AE profiles to those previously described in cancers arising from viral or chemical etiologies.     

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.     

Mitochondrial superoxide decreases yeast survival in stationary phase

Yeast lacking mitochondrial superoxide dismutase (MnSOD) display shortened stationary-phase survival and provide a good model system for studying mitochondrial oxidative damage. We observed a marked decrease in respiratory function preceding stationary-phase death of yeast lacking MnSOD (sod2Delta). Agents (mitochondrial inhibitors) that are known to increase or decrease superoxide production in submitochondrial particles affected stationary-phase survival in a manner inversely correlated with their effects on superoxide production, implicating superoxide in this mitochondrial disfunction. Similar but less-dramatic effects were observed in wild-type yeast. The activities of certain mitochondrial enzymes were particularly affected. In sod2Delta yeast the activity of aconitase, a 4Fe-4S-cluster-containing enzyme located in the matrix, was greatly and progressively decreased as the cells established stationary phase. Succinate dehydrogenase activity also decreased in MnSOD mutants; cytochrome oxidase and ATPase activities did not. Aconitase could be reactivated by addition of materials required for cluster assembly (Fe3+ and a sulfur source), both in extracts and in vivo, indicating that inactivation of the enzyme was by disassembly of the cluster. Our results support the conclusion that superoxide is generated in the mitochondria in vivo and under physiological conditions and that MnSOD is the primary defense against this toxicity. When the balance between superoxide generation and MnSOD activity is disrupted, superoxide mediates iron release from mitochondrial iron-sulfur clusters, leading first to loss of mitochondrial function and then to death, independently of mtDNA damage. These results raise the possibility that similar processes may occur in higher eukaryotes.     

Studies on superoxide dismutase activities in virulent and avirulent strains of Agrobacterium tumefaciens and also in normal and crown gall tumor cells of Bryophyllum calycinum

Superoxide dismutase activity in virulent strains of Agrobacterium tumefaciens was found to be higher than that in avirulent strains. Polyacrylamide gel electrophoresis revealed two isoenzymes in both these strains. These isoenzymes are suggested to be iron and manganese containing superoxide dismutases. Crown gall tumor cells of the plant Bryophyllum calycinum were found to have higher superoxide dismutase activity than the normal plant cells. Polyacrylamide gel electrophoresis revealed two isoenzymes in both normal and crown gall tumor cells. Advantages of the higher superoxide dismutase activities in respect of the survival of virulent strains of A. tumefaciens and crown gall tumor growth have been discussed.     

Subcellular distribution of superoxide dismutases in human neutrophils. Influence of myeloperoxidase on the measurement of superoxide dismutase activity

We have identified two distinct pools of superoxide dismutase in fractions of human peripheral neutrophils obtained by the isopycnic fractionation of homogenates of the latter with linear sucrose gradients. Superoxide dismutase activity, observed with polyacrylamide gels impregnated with Nitro Blue Tetrazolium, was present in: (1) the mitochondrial fraction [density (rho) 1.169g/ml], containing the high-molecular-weight KCN-resistant enzyme, and (2) the cytoplasm fraction, containing the low-molecular-weight KCN-sensitive enzyme. Superoxide dismutase activity, observed with a quantitative assay involving cytochrome c, was present in: (1) the mitochondria, (2) the cytoplasm, and (3) the azurophil-granule fractions (rho=1.206 and 1.222g/ml). No substantial enzyme activity was observed in specific-granule fractions (rho=1.187g/ml) or in the membranous fraction (rho=1.136g/ml) in either assay. The apparent superoxide dismutase activity observed in the azurophil granules with the cytochrome c assay was attributable not to true superoxide dismutase but to myeloperoxidase, an enzyme found solely in the azurophil granules. In the presence of H(2)O(2), human neutrophil myeloperoxidase oxidized ferrocytochrome c. Thus, in the cytochrome c assay for superoxide dismutase, the oxidation of ferrocytochrome c by myeloperoxidase mimicked the inhibition of reduction of ferricytochrome c by superoxide dismutase. When myeloperoxidase was removed from azurophilgranule fractions by specific immuno-affinity chromatography, both myeloperoxidase and apparent superoxide dismutase activities were removed. It is concluded that there is no detectable superoxide dismutase in either the azurophil or specific granules of human neutrophils. Mitochondrial superoxide dismutase, 15% of the total dismutase activity of the cells, occurred only in fractions of density 1.160g/ml, where isocitrate dehydrogenase and cytochrome oxidase were also observed.     

利用酿酒酵母转座子文库筛选MTM1基因缺失表型相关基因

【目的】MTM1基因对于维持锰超氧化物歧化酶的活性和线粒体正常功能十分重要,MTM1基因的缺失会严重影响锰超氧化物歧化酶活性,并损伤线粒体功能,使酵母在非发酵培养基上不能生长。为加深对MTM1基因功能及其相关基因的研究,尝试利用转座子文库筛选MTM1基因缺失表型相关基因,寻找哪些位置的转座子插入能挽救MTM1基因缺失导致的生长缺陷。【方法】因MTM1基因的缺失造成的损伤不可逆,直接转入文库无法筛选得到MTM1基因缺失表型相关基因,本研究利用外源MTM1基因菌株和mTn-lacZ/LEU2酿酒酵母转座子文库进行筛选,寻找能挽救mtm1突变体生长缺陷的转座子插入位点。【结果】发现转座子插入HSL1和TPS2基因能挽救mtm1突变体的生长缺陷。【结论】我们的结果为深入了解MTM1基因的功能提供了线索。     

Localization, isolation and characterization of Mn-superoxide dismutase in bovine adrenocortical cells

Cyanide-insensitive superoxide dismutase activity was present in both cytosol (26%) and mitochondrial (64%) fractions of bovine adrenal cells. The cyanide-insensitive superoxide dismutase was isolated from the mitochondrial fraction. It contained 2.2 g atoms of manganese per mol of enzyme. The enzyme had a molecular weight of 82,000 and a subunit molecular weight of 22,000. The isoelectric point, amino acid composition, and spectra of visible and ultraviolet regions were similar to those of the Mn-superoxide dismutase from the other mitochondria.     

Mitochondrial adaptations to obesity-related oxidant stress

It is not known why viable hepatocytes in fatty livers are vulnerable to necrosis, but associated mitochondrial alterations suggest that reactive oxygen species (ROS) production may be increased. Although the mechanisms for ROS-mediated lethality are not well understood, increased mitochondrial ROS generation often precedes cell death, and hence, might promote hepatocyte necrosis. The aim of this study is to determine if liver mitochondria from obese mice with fatty hepatocytes actually produce increased ROS. Secondary objectives are to identify potential mechanisms for ROS increases and to evaluate whether ROS increase uncoupling protein (UCP)-2, a mitochondrial protein that promotes ATP depletion and necrosis. Compared to mitochondria from normal livers, fatty liver mitochondria have a 50% reduction in cytochrome c content and produce superoxide anion at a greater rate. They also contain 25% more GSH and demonstrate 70% greater manganese superoxide dismutase activity and a 35% reduction in glutathione peroxidase activity. Mitochondrial generation of H(2)O(2) is increased by 200% and the activities of enzymes that detoxify H(2)O(2) in other cellular compartments are abnormal. Cytosolic glutathione peroxidase and catalase activities are 42 and 153% of control values, respectively. These changes in the production and detoxification of mitochondrial ROS are associated with a 300% increase in the mitochondrial content of UCP-2, although the content of beta-1 ATP synthase, a constitutive mitochondrial membrane protein, is unaffected. Supporting the possibility that mitochondrial ROS induce UCP-2 in fatty hepatocytes, a mitochondrial redox cycling agent that increases mitochondrial ROS production upregulates UCP-2 mRNAs in primary cultures of normal rat hepatocytes by 300%. Thus, ROS production is increased in fatty liver mitochondria. This may result from chronic apoptotic stress and provoke adaptations, including increases in UCP-2, that potentiate necrosis.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Catalase and Superoxide Dismutase of Root-Colonizing Saprophytic Fluorescent Pseudomonads

Root-colonizing, saprophytic fluorescent pseudomonads of the Pseudomonas putida-P. fluorescens group express similar levels of catalase and superoxide dismutase activities during growth on a sucrose- and amino acid-rich medium. Increased specific activities of catalase but not superoxide dismutase were observed during growth of these bacteria on components washed from root surfaces. The specific activities of both enzymes were also regulated during contact of these bacteria with intact bean roots. Increased superoxide dismutase and decreased catalase activities were observed rapidly, by 10 min upon inoculation of cells onto intact bean roots. Catalase specific activity increased with time to peak at 12 h before declining. By 48 h, the cells displayed this low catalase but maintained high superoxide dismutase specific activities. Catalase with a low specific activity and a high superoxide dismutase activity also were present in extracts of cells obtained from 7-day-old roots colonized from inoculum applied to seed. This specific activity of superoxide dismutase of root-contacted cells was about fourfold-higher in comparison to cells grown on rich medium, whereas the specific activity for catalase was reduced about fivefold. A single catalase isozyme, isozyme A, and one isozyme of superoxide dismutase, isozyme 1, were detected during growth of the bacteria on root surface components and during exposure of cells to intact bean roots for 1 h. An additional catalase, isozyme B, was detected from bacteria after exposure to the intact bean roots for 12 h. Catalase isozyme A and superoxide dismutase isozyme 1 were located in the cytoplasm and catalase band B was located in the membrane of P. putida.     

Antioxidative defense to lead stress in subcellular compartments of pea root cells

Lead, similar to other heavy metals and abiotic factors, causes many unfavorable changes at the subcellular and molecular levels in plant cells. An increased level of superoxide anion in Pisum sativum root cells treated with 1 mM Pb(NO3)2 evidenced oxidative stress conditions. We found increased activities of enzymatic components of the antioxidative system (catalase and superoxide dismutase) in the cytosol, mitochondrial and peroxisomal fractions isolated from root cells of Pisum sativum grown in modified Hoagland medium in the presence of lead ions (0.5 or 1 mM). Two isoenzyme forms of superoxide dismutase (Cu,Zn-SOD and Mn-SOD) found in different subcellular compartments of pea roots were more active in Pb-treated plants than in control. Increased amount of alternative oxidase accompanied by an increased activity of this enzyme was found in mitochondria isolated from lead-treated roots. These results show that plants storing excessive amounts of lead in roots defend themselves against the harmful oxidative stress caused by this heavy metal.     

Human aging brain disorders: Role of antioxidant enzymes

In order to investigate the role of two free radical detoxificant enzymes in patients with aging brain disorders, superoxide dismutase (SOD) and catalase (CAT) activities have been measured in blood from male and female human patients of different ages with several types of aging brain disorders. When compared with activities in the normal population, we have detected: 1) SOD and CAT activities are decreased in patients with Parkinson disease. 2) SOD activity seems to be normal and CAT activity is decreased in patients with dementia. 3) In the patients with stroke, SOD activity is normal, while CAT activity is decreased. SOD activity was measured in red blood cells using the Minami and Yoshikawa method. CAT activity was measured in hemolysates by the method of Aebi. We can conclude that SOD and CAT activities in patients with Parkinson disease are decreased.     

Superoxide dismutase and peroxidase: a positive activity stain applicable to polyacrylamide gel electropherograms.

The oxidation of dianisidine, photosensitized by riboflavin, is accelerated by superoxide dismutase. Polyacrylamide gel electropherograms soaked in riboflavin plus dianisidine and subsequently illuminated develop stable brown bands at positions bearing superoxide dismutase activity. This constitutes a new, convenient, and advantageous activity stain for this class of enzymes. Peroxidases are also stained by this procedure due to the photochemical production of H₂O₂. This does not constitute an interference with the specificity of the stain, since peroxidase bands develop more slowly than superoxide dismutase bands and can be further identified through the use of inhibitors or of independent staining for peroxidase. The new, positive activity stain for superoxide dismutases can be applied to crude extracts of cells.     

Manganese superoxide dismutase regulation and cancer

Mitochondria are the power plants of the eukaryotic cell and the integrators of many metabolic activities and signaling pathways important for the life and death of a cell. Normal aerobic cells use oxidative phosphorylation to generate ATP, which supplies energy for metabolism. To drive ATP production, electrons are passed along the electron transport chain, with some leaking as superoxide during the process. It is estimated that, during normal respiration, intramitochondrial superoxide concentrations can reach 10^?12 M. This extremely high level of endogenous superoxide production dictates that mitochondria are equipped with antioxidant systems that prevent consequential oxidative injury to mitochondria and maintain normal mitochondrial functions. The major antioxidant enzyme that scavenges superoxide anion radical in mitochondria is manganese superoxide dismutase (MnSOD). Extensive studies on MnSOD have demonstrated that MnSOD plays a critical role in the development and progression of cancer. Many human cancer cells harbor low levels of MnSOD proteins and enzymatic activity, whereas some cancer cells possess high levels of MnSOD expression and activity. This apparent variation in MnSOD level among cancer cells suggests that differential regulation of MnSOD exists in cancer cells and that this regulation may be linked to the type and stage of cancer development. This review summarizes current knowledge of the relationship between MnSOD levels and cancer with a focus on the mechanisms regulating MnSOD expression.