Superoxide dismutase in ruminal bacteria.

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.     

Factors Related to the Oxygen Tolerance of Anaerobic Bacteria

The effect of atmospheric oxygen on the viability of 13 strains of anaerobic bacteria, two strains of facultative bacteria, and one aerobic organism was examined. There were great variations in oxygen tolerance among the bacteria. All facultative bacteria survived more than 72 h of exposure to atmospheric oxygen. The survival time for anaerobes ranged from less than 45 min for Peptostreptococcus anaerobius to more than 72 h for two Clostridium perfringens strains. An effort was made to relate the degree of oxygen tolerance to the activities of superoxide dismutase, catalase, and peroxidases in cell-free extracts of the bacteria. All facultative bacteria and a number of anaerobic bacteria possessed superoxide dismutase. There was a correlation between superoxide dismutase activity and oxygen tolerance, but there were notable exceptions. Polyacrylamide gel electropherograms stained for superoxide dismutase indicated that many of the anaerobic bacteria contained at least two electrophoretically distinct enzymes with superoxide dismutase activity. All facultative bacteria contained peroxidase, whereas none of the anaerobic bacteria possessed measurable amounts of this enzyme. Catalase activity was variable among the bacteria and showed no relationship to oxygen tolerance. The ability of the bacteria to reduce oxygen was also examined and related to enzyme content and oxygen tolerance. In general, organisms that survived for relatively long periods of time in the presence of oxygen but demonstrated little superoxide dismutase activity reduced little oxygen. The effects of medium composition and conditions of growth were examined for their influence on the level of the three enzymes. Bacteria grown on the surface of an enriched blood agar medium generally had more enzyme activity than bacteria grown in a liquid medium. The data indicate that superoxide dismutase activity and oxygen reduction rates are important determinants related to the tolerance of anaerobic bacteria to oxygen.     

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.     

Tobacco nectarin I. Purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defensE of floral reproductive tissues

Nectarin I, a protein that accumulates in the nectar of Nicotiana sp. , was determined to contain superoxide dismutase activity by colorimetric and in-gel assays. This activity was found to be remarkably thermostable. Extended incubations at temperatures up to 90 degrees C did not diminish the superoxide dismutase activity of nectarin I. This attribute allowed nectarin I to be purified to homogeneity by heat denaturation of the other nectar proteins. By SDS-polyacrylamide gel electrophoresis, nectarin I appeared as a 29-kDa monomer. If the protein sample was not boiled prior to loading the gel, then nectarin I migrated as 165-kDa oligomeric protein. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the protomer subunit was found to be a 22.5-kDa protein. Purified nectarin I contained 0.5 atoms of manganese/monomer, and the superoxide dismutase activity of nectarin I was not inhibited by either H(2)O(2) or NaCN. Following denaturation, the superoxide dismutase activity was restored after Mn(2+) addition. Addition of Fe(2+), Cu(2+), Zn(2+), and Cu(2+)/Zn(2+) did not restore superoxide dismutase activity. The quaternary structure of the reconstituted enzyme was examined, and only tetrameric and pentameric aggregates were enzymatically active. The reconstituted enzyme was also shown to generate H(2)O(2). Putative nectarin I homologues were found in the nectars of several other plant species.     

Scavenging of superoxide and hydrogen peroxide in blue‐green algae (cyanobacteria)

Blue‐green algae (cyanobacteria) have evolved as the most primitive, oxygenic, plant‐type photosynthetic organisms. Within a single prokaryotic cell, they have uniquely accommodated both oxygenic photosynthesis and aerobic respiration, which are known to produce superoxide and hydrogen peroxide as inevitable byproducts. Two types of superoxide dismutase have been characterized in both N₂‐fixing and non‐N₂‐fixing cyanobacteria, namely cytosolic iron‐containing superoxide dismutase and thylakoid‐bound manganese‐containing superoxide dismutase. No qualitative differences between various cell types (vegetative cells, heterocysts) were found. In contrast to chloroplasts, most of the cyanobacterial species show catalatic activity. From two species the corresponding enzymes have been characterized as typical prokaryotic (bifunctional) catalase‐peroxidases with homologies to cytochrome c peroxidases and ascorbate peroxidases. In addition to catalatic activity, some strains exhibit ascorbate peroxidase activity, but to date there are no reports detailing purification and characterization.
Cyanobacteria were found to contain low intracellular ascorbate concentrations (30‐100 µ M ) and 2‐5 m M glutathione. Both monodehydroascorbate and glutathione reductase activities were detected in most species examined, whereas dehydroascorbate reductase activity was absent. The question as to whether a glutathione‐ascorbate cycle exists in cyanobacteria cannot be answered at present.     

Analytical subcellular fractionation of rabbit alveolar macrophages with particular reference to the subcellular localisation of pyridine nucleotide-dependent superoxide-generating systems and superoxide dismutase

Normal and Bacillus Calmette-Guerin (BCG) vaccine-induced rabbit alveolar macrophage homogenates were fractionated by isopycnic density gradient centrifugation. Superoxide dismutase-inhibitable NAD(P)H-dependent nitro-blue tetrazolium reductase was found localised to endoplasmic reticulum and mitochondria. The normal macrophages tended to contain more of this activity than the BCG-induced macrophages. Two superoxide dismutases were found: cyanide-sensitive superoxide dismutase was predominantly present in the cytosol, with a small proportion in mitochondria; cyanide-resistant superoxide dismutase was found confined to mitochondria. Neither differed in specific activity betw-en the normal and BCG-induced macrophages.     

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.     

Anti-oxidant defense of the cell Desulfovibrio desulfuricans B-1388

The extracts of Desulfovibrio desulfuricans B-1388 cells, grown in anaerobic condition, display the superoxide dismutase activity. The maximum value of level activity (1.02 E/min/mg) is observed in the stationary phase of growth. Essentially the whole enzyme is localized in periplasmic fraction. Cells Desulfovibrio desulfuricans B-1388 do not show the catalase activity but contain active NADH- and NADPH-peroxidases. The activity of involved peroxidases depends on the physiological condition of culture.     

Histochemistry of reactive oxygen-species (ROS)-generating oxidases in cutaneous and mucous epithelia of laboratory rodents with special reference to xanthine oxidase

Cutaneous and mucous epithelia of various organs of laboratory rodents were analysed histochemically for reactive oxygen species (ROS)-generating oxidases using cerium methods. High activities of xanthine oxidase and also superoxide dismutase were present in orthokeratotic stratified squamous epithelia of skin, lips, esophagus and forestomach and parakeratotic keratinizing stratified epithelia of vagina, tongue and penis. Moreover, activity was found in simple epithelium of the uterus and intestine of rats, mice and guinea-pigs. Moderate activities of monoamine oxidase and D-amino acid oxidase were only seen in enterocytes of large and small intestine, whereas alpha-hydroxy acid oxidase could not be detected at all. With the use of specific inhibitors for superoxide anions-producing xanthine oxidase and H2O2-generating superoxide dismutase it was shown that epithelial cells of all studied external and internal surface epithelia contain a highly effective xanthine oxidase-superoxide dismutase system. It is hypothesized that this system might have a general microbicidal function and might play a special role in tumor promotion of the skin.     

Reactive oxygen species are partially involved in the bacteriocidal action of hypochlorous acid.

Hypochlorous acid (HOCl) is probably the most widely used disinfectant worldwide and has an important role in inflammatory reaction and in human resistance to infection. However, the nature and mechanisms of its bactericidal activity are still poorly understood. Bacteria challenged aerobically with HOCl concentrations ranging from 9.5 to 76 microM exhibit higher ability to form colonies anaerobically than aerobically. Conversely, aerobic plating greatly increased lethality after an anaerobic HOCl challenge, although anaerobic survival did not depend on whether HOCl exposure was aerobic or anaerobic. Even a short transient exposure to air after anaerobic HOCl challenge reduced anaerobic survival, indicative of immediate deleterious effects of oxygen. Exposure to HOCl can cause lethal DNA damage as judged by the fact that recA sensitivity to HOCl was oxygen dependent. Antioxidant defenses such as reduced glutathione and glucose-6-phosphate dehydrogenase were depleted or inactivated at 10 microM HOCl, while other activities, such as superoxide dismutase, dropped only above 57 microM HOCl. Cumulative deficiencies in superoxide dismutase and glucose-6-phosphate dehydrogenase rendered strains hypersensitive to HOCl. This indicates that part of HOCl toxicity on Escherichia coli is mediated by reactive oxygen species during recovery.     

Antioxidative enzymes of sulfate-reducing bacterium desulfovibrio desulfuricans: superoxide dismutase and peroxidases

Extracts of Desulfovibrio desulfuricans B-1388 cells grown under anaerobic conditions displayed superoxide dismutase activity. The maximal activity was found during the stationary growth phase. The enzyme was virtually completely located in the periplasm fraction. D. desulfuricans B-1388 lacked catalase activity but contained active NADH- and NADPH-peroxidases. The activity of NADH-peroxidase depended on the physiological state of the culture. On changing the growth conditions (the presence of 5% CO in the gaseous phase), the activity of superoxide dismutase decreased.     

Chemical and physical differentiation of superoxide dismutases in anaerobes.

Superoxide dismutase activity in crude or partially purified cell extracts from several species and strains of obligate anaerobe Bacteroides was inhibited instantaneously by NaN3 and was inactivated rapidly upon incubation with H2O2. The extent of NaN3 inhibition varied from 41 to 93%, and the half-life of the enzymatic activity in 5 mM H2O2 ranged from 1.2 to 6.1 min, depending upon the organism tests. When grown in a defined medium containing 59Fe, Bacteroides fragilis (VPI 2393) incorporated radiolabel into a 40,000-molecular-weight NaN3- and H2O2-sensitive superoxide dismutase but did not incorporate 54Mn into that protein under similar growth conditions. The anaerobe Actinomyces naeslundii (VPI 9985) incorporated 54Mn but not 59Fe into a NaN3-insensitive and H2O2-resistant superoxide dismutase. The apparent molecular weight of the superoxide dismutase from this and several other Actinomyces spp. was estimated to be 110,000 to 140,000. Comparison of these data with studies of homogeneous metallosuperoxide dismutases suggests that the Bacteroides spp. studied contain a ferrisuperoxide dismutase, whereas Actinomyces spp. contain a managanisuperoxide dismutase.     

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.     

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.     

Characterization of a superoxide dismutase gene from the archaebacterium Methanobacterium thermoautotrophicum

A gene encoding superoxide dismutase (SOD) was cloned from the archaebacterium Methanobacterium thermoautotrophicum, the first example from an anaerobic bacterium. The deduced amino acid sequence showed overall similarity to sequences of known Mn- and Fe-SODs from aerobic organisms. Judging from a detailed sequence comparison, the cloned SOD gene is classified as Mn-SOD. By comparison of Mn-SOD sequences among various species it was suggested that archaebacterial superoxide dismutase is a direct descendant of a primordial enzyme. Between a putative promoter and the start codon there is an inverted repeat sequence which is also found in the counterpart of Halobacterium halobium.     

CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in glutathione-deficient human fibroblasts

The effect of genetically determined glutathione deficiency on the fibroblast content of CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase was investigated. No significant differences between glutathione-deficient and -proficient human fibroblasts were revealed. There was a large variation in the content of the investigated enzymes in fibroblasts grown and analysed on different occasions. Whereas the contents of CuZn superoxide dismutase, catalase and glutathione peroxidase did not deviate much from what has been found in other human cell-lines and tissues, the fibroblasts were found to contain exceptional amounts of Mn superoxide dismutase.     

Catalase, superoxide dismutase, and the production of O2-sensitive mutants of Bacillus coagulans

A number of facultatively anaerobic members of the genus Bacillus were screened for their catalase, diaminobenzidine peroxidase, and superoxide dismutase activities. A strain of Bacillus coagulans (7050) lacking peroxidatic activity and containing single catalatic and superoxide dismutase activities was selected. Responses of the superoxide dismutase activity and catalase level to the partial pressure of oxygen, and Fe and Mn levels, as well as to aerobic and fermentative metabolism, were determined. There appeared to be a relationship between high endogenous catalase levels and the high H2O2 evolution and KCN insensitivity of B. coagulans respiration. Bacillus coagulans 7050 was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and screened for the expression of oxygen intolerance. All of the 38 stable oxygen sensitive mutants obtained had very low or completely absent catalatic activity and catalase protein. No mutant lacked superoxide dismutase, although five showed significantly lowered levels of the enzyme. Exogenous bovine liver catalase restored aerotolerance and reduced cell pleomorphism in the mutants.     

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.     

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.