Superoxide dismutase, glutathione peroxidase and catalase in developing rat brain.

The specific activities of Cu,Zn- and Mn-superoxide dismutases, of glutathione peroxidase and of catalase, the enzymes considered to be specifically involved in the defence of the cell against the partially reduced forms of oxygen, were determined as the function of postnatal age in the early (up to 60 days) period of rat brain development. The enzymes were assayed in the cytoplasmic fraction, in the crude mitochondrial fraction including peroxisomes, and in the mitochondria. The results show that the temporal changes of these enzymes cannot be correlated with each other, thus indicating that they do not concertedly parallel the increasing activity of aerobic brain metabolism during development. Specifically the cytoplasmic fraction shows a gradual increase of the Cu,Zn-superoxide dismutase activity with age, whereas the glutathione peroxidase activity is constant from birth. Furthermore the increase of the mitochondrial Mn-superoxide dismutase as a function of postnatal age is more remarkable than that of the cytoplasmic Cu,Zn-enzyme. Higher activities of catalase in adult animals are detectable only in the subcellular fraction containing peroxisomes, because of the modest catalase activity of the brain. These results indicate independent regulation of the expression of these enzyme activities in the process of brain differentiation and point to a relative deficiency of enzymic protection of the brain differentiation and point to a relative deficiency of enzymic protection of the brain against potentially toxic oxygen derivatives. This situation is similar to the pattern already described in the rat heart and in rat and mouse ascites-tumour cells, at variance with the much more efficient enzyme pattern present in rat hepatocytes.     

Isolation and characterization of basic superoxide dismutase consisting of Mr-25,000 subunits in rat liver

1. A basic protein (pI = 9.0) exhibiting superoxide dismutase activity was purified to homogeneity from rat liver by DEAE-cellulose, CM-cellulose and S-hexylglutathione affinity gel chromatography, chromatofocusing and Sephadex G-150 gel filtration. 2. The purified enzyme had specific activity of 4700 units/mg protein. The activity was not affected by 2 mM KCN. Manganese was detected in the enzyme preparation; the content was 0.9 mol/mol subunit. The N-terminal sequence of the first 23 amino acids of the enzyme exhibited a strong homology (except at position 11) with the mature protein of human Mn-superoxide dimutase. It is, therefore, concluded that the purified enzyme is Mn-superoxide dismutase. 3. The N-terminal amino acid sequence showed that about 50% of tyrosine at position 11 was substituted by glutamine, suggesting the existence of microheterogeneity of the superoxide dismutase protein. 4. The superoxide dismutase purified here was found to consist of subunits with an apparent relative molecular mass of 25,000. This larger than the value hitherto reported for rat liver Mn-superoxide dismutase (Mr 2,400); the previous low value is attributed to differences in methods. 5. The enzyme was shown by immuno-blotting to be exclusively localized in the mitochondrial fraction in the liver. The tissue content of Mn-superoxide dismutase is organ-specific, and was the highest in heart. The precursor protein of the Mn-superoxide dismutase was not detectable in the liver cytosolic and mitochondrial fractions as well as in several extrahepatic organs (lung, heart, brain, muscle, kidney and testis), suggesting rapid transport across mitochondrial membranes and processing of the superoxide dismutase protein.     

Determination of Manganese Superoxide Dismutase Activity By Direct Spectrophotometry

A method to determine Mn-superoxide dismutase activity by measuring directly the rate of decay of O₂^- in a spectrophotometer, is described. Decay of O₂^- generated by KO₂ at pH 9.5, was monitored as the fall in absorbance (A_250nm-A_360nm). Mn-superoxide dismutase was determined as the activity of cyanide-resistant superoxide dismutase, calculated from the rate of O₂^- dismutation. Mn-superoxide dismutase could be determined in the presence of a 700 times higher Cu, Zn-superoxide dismutase activity. The alkaline pH did not cause analytical problems. The assay was used to measure both Mn- and Cu, Zn-superoxide dismutase activity in mitochondrial preparations. The assay had a detection limit of 2.8 ng/ml when Mn-superoxide dismutase from E. coli was used, and the between-day CV was 5.8%. The assay is an alternative to indirect methods for detecting superoxide dismutase activity.     

Comparative study of superoxide dismutase and glutathione peroxidase activity in embryos and skeletal muscles of adult fish

The activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase in loach and sturgeon embryogenesis as well as in red and white skeletal muscles of loach was studied. The specific activity of cytoplasmic and mitochondrial forms of superoxide dismutase in developing sturgeon embryos was higher than in loach embryos, which may be due to oxygen conditions under which these species develop in nature. A similar dependence was also observed for the activity of glutathione peroxidase in embryos of these fish species. A comparative study of specific superoxide dismutase activity in loach and sturgeon embryos and in loach skeletal muscles showed that the activity of cytoplasmic superoxide dismutase is maximum in red and white muscles and minimum in loach embryos, whereas the activity of the mitochondrial form of this enzyme is maximum in red skeletal muscles.     

Intracellular distribution of creatine kinase isoenzymes in the brains and hearts of rats at different stages of postnatal development]

The total activity and range of the creatine kinase (CK) isozymes have been studied in the homogenate and subcellular fractions (nuclei, mitochondria, cytoplasm) of the rat brain and heart during postnatal ontogenesis. The total activity of CK in the brain and heart of newborn rats was found to be 4 and 2 times less, resp., than in those of adults. The age patterns were established in the activity of cytoplasmic (CK-1, CK-2 and CK-3) and mitochondrial (CK-4) isozymes. During the whole postnatal development the rat brain contains only one cytoplasmic isozyme, CK-1. In the heart of newborn rats, as compared with adults, the content of CK-1 and CK-2 is much higher and that of CK-3 lower. On the 12-15th day of life the range of the CK isozymes approaches that characteristic of adult animals. The activity of CK-4 was found in the brain on the 5-7th day of life and in the heart on 12-15th day. In the range of the CK isozymes in the adult brain the content of mitochondrial CK amounts to 19.3% and in the heart to 16.5%. The data obtained complement the literary ones suggesting the low level of energy-forming processes in the brain and heart cells at the early stages of the rat postnatal development.     

Increased Superoxide Dismutase Activity in Aged Human Cerebrospinal Fluid and Rat Brain Determined by Electron Spin Resonance Spectrometry Using the Spin Trap Method

Superoxide dismutase (SOD) activity in CSF of patients was determined by electron spin resonance spectrometry using the spin trap method. Variation in SOD activity was found among patients. SOD activity in CSF of subjects increased with age and this was identified as Cu,Zn-SOD activity by electrophoresis. In addition, animal experiments showed that SOD activities were higher in mitochondrial and cytosol fractions of aged rats than in those of adult rats. This finding on aged rat brain validates the increase of SOD activity in aged human CSF.     

Fractionation of specific mitochondrial and cytoplasmic tRNAs obtained from calf brain.

—Total tRNA fractions were isolated from pure mitochondrial and cytoplasmic calf brain preparations. After incubation with homologous crude preparations of aminoacyl-tRNA ligases in the presence of [¹⁴C]-glutamic acid, tRNAs were separated chromatographically on BD-cellulose columns and in reversed phase chromatography systems. In both of the methods used, cytoplasmic tRNA preparations revealed a larger number of radioactivity peaks. In experiments with double labelling, five radioactivity peaks for cytoplasmic glutamyl-tRNAs corresponded to only three mitochondrial glutamyl-tRNA fractions. The results imply the presence of isoaccepting species of tRNA in brain.     

Comparison of cytoplasmic superoxide dismutase in liver,heart and brain of aging rats and mice

Comparisons of the specific activity of cytoplasmic superoxide dismutase, in homogenates of liver, brain and heart demonstrate considerably reduced activity in livers of aging rats and mice, a very small reduction in specific activity in the heart and no reduction in the brain of aging animals.Antisera elicited in rabbits against purified superoxide dismutase from liver of either young or old rats revealed complete cross-reactivity with the heart and brain enzymes. They also exhibited complete cross-reactivity with the mouse enzymes from all three organs. This finding has, for the first time, enabled a comparison of possible age-dependent alterations in the same enzyme antigen in different cell types.Despite the differences in age-related changes in specific activity in homogenates of liver, heart and brain the enzyme shows a considerable decline in catalytic activity per antigenic unit in $\text{all}$ three organs in both aging rats and mice.     

Differential temperature sensitivity of pea superoxide dismutases

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

The appearance, properties, and functions of gluconeogenic enzymes in the liver and kidney of the guinea pig during fetal and early neonatal development.

The changes in the activity and properties of the four gluconeogenic enzymes have been followed during development of the guinea pig. Pyruvate carboxylase was almost exclusively mitochondrial and kinetically identical to the adult liver enzyme and did not appear in significant activity until after day 50 when it rose to values several times higher than those in the adult liver, then fell after birth. Little activity was detected in the fetal kidney. Phosphoenolpyruvate carboxylase appeared in the fetal liver from day 30 on, both in the mitochondrial and cytoplasmic fractions. The cytoplasmic enzyme was kinetically and chromatographically identical to the mitochondrial enzyme of the fetal and maternal liver. After birth the activity of the cytoplasmic enzyme increased and that of the particulate enzyme fell. Fetal kidney activity appeared several days before birth. Fructose 1,6-diphosphatase and glucose 6-phosphatase appeared in the fetal liver and kidney after day 40; the former showed no postnatal change while the latter rose 10-fold after birth. Fetal liver fructose 1,6-diphosphatase was more sensitive to AMP and fructose 1,6-diphosphate inhibition but was chromatographically indistinguishable from the maternal liver enzyme. Despite the presence of the gluconeogenic enzymes, gluconeogenesis and glyconeogenesis were not detected in the fetal liver until 7–9 days before birth. While the synthesis of glyceride-glycerol from 3-carbon compounds was detected from 35–40 days onwards and some of the gluconeogenic enzymes participate in that pathway, gluconeogenesis was not detected in the fetal kidney.     

Disordered functioning of the superoxide radical-superoxide dismutase system in rat liver ischemia

The rate of O2 radical generation in microsomal membranes (VO2), the activity of cytosol superoxide dismutase (Cu, ZnSOD) and mitochondrial superoxide dismutase (MnSOD), and the activity of xanthine oxidizing system (XO) after a two-hour ischemia following a 24-hour reoxygenation of the rat liver were investigated. The high value of VO2, as compared to Cu, ZnSOD activity, may result in regulation disorders in O2-SOD system during ischemia. During reoxygenation, xanthine oxidizing system in combination with lowered Cu, ZnSOD activity may substantially contribute to the disturbance.     

Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging

The activities of Cu,Zn superoxide dismutase, glutathione peroxidase, catalase and glutathione reductase in neuronal and glial cell-enriched fractions obtained from the cerebral cortex of rat brain during aging (15, 30, 90, 350, 750 days of age) were assayed. Our results showed that glutathione peroxidase, catalase and glutathione reductase activities varied little during the examined periods. Only the Cu,Zn superoxide dismutase activity decreased notably from 15th to 750th day of age in both neuronal and glial cells, moreover the activities of all enzymes studied were always detected at lower levels in neuronal cells with respect to glial cells. In agreement with diminished SOD activity, the lipid peroxidation showed an elevated increase with aging; this fact is more evident in neuronal than in glial cells. In conclusion our data show that Cu,Zn superoxide dismutase is the most affected antioxidant enzymatic system of brain aging and it could be responsible for the increased lipid peroxidation in both cell types examined.A preliminary report of these results was presented at the 19th Meeting F.E.B.S. Rome July 2–7, 1989.     

Variation of superoxide dismutases during the development of the fruit fly Ceratitis capitata L.

Superoxide dismutase levels were estimated in eggs, larvae and pupae of the fruit fly $\text{Ceratitis capitata}$, as well as in adult flies. No changes occur in the first three stages, but development of the adult fly is accompanied by a large increase in mitochondrial superoxide dismutase per gm of material, and a much smaller relative increase in the cytoplasmic enzyme.     

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.     

Effect of chronic ethanol consumption on emotional stress in the white rat

Chronic pain emotional stress (PES), paired action of the white noise and electric skin stimulation and chronic (during 7 months) ethanol consumption in white rats were shown to act in the same direction. Hypertension, decrease of respiratory rate and increase of Hildebrandt index were observed as a result of PES, ethanol consumption, and especially under PES during ethanol consumption. Ethanol consumption by the animals led to their growth retardation and increase of the spleen and heart mass. Accidental thymus involution was noted both under ethanol consumption and PES. Activation of lipid peroxidation and decrease of superoxide dismutase activity (of its mitochondrial form especially) as well as of Na+,K+-ATP-ase activity were observed in brain homogenates of the rats after PES, while the general ATP-ase activity remained unchanged. An increase of triiodothyronine level and the tendency to thyroxine level increase as well as a decrease of superoxide dismutase activity were observed in the blood serum of these animals. A tendency towards lipid peroxidation level decrease and to brain superoxide dismutase activity increase, as well as blood antioxidation activity increase (evaluated by transferrin and coeruloplasmin contents and by serum superoxide dismutase activity) and a decrease of thyroxine level were observed as a result of ethanol consumption. The mechanisms are discussed of the "anti-stress" action of short-term ethanol consumption and of the action of its chronic consumption, additive to PES.     

Specific induction of indoleamine 2,3-dioxygenase by bacterial lipopolysaccharide in the mouse lung

Indoleamine 2,3-dioxygenase activity in the supernatant fractions (30,000g, 30 min) from various tissues of mice increased almost linearly after a single intraperitoneal administration of bacterial lipopolysaccharide (5 to 20 μg/mouse). The most prominent effect was observed in the lung, where both specific and total enzyme activities increased 40 to 80-fold during the first 24 h. Significant (10- to 20-fold) stimulation was also observed in the seminal vesicle, coagulating gland, colon, and caecum, and severalfold in the trachea, stomach, heart, small intestine, and spleen. Lipid A fraction, the biologically active unit in the lipopolysaccharide complex, was as active as the lipopolysaccharide preparations from either Escherichia coli or Salmonella S and R mutant strains, whereas the polysaccharide fraction was inactive under identical experimental conditions. When mice were pretreated with a series of daily injections of bacterial lipopolysaccharide, enzyme induction was no longer evident, indicating that tolerance to this agent had developed and that enzyme induction was caused by lipopolysaccharide but not by possible contaminants in the preparations. The enzyme activities from normal and lipopolysaccharide-treated mice were exclusively found in the soluble fractions of mouse lung homogenates. Other enzyme activities in the lung such as lysosomal (acid phosphatase), microsomal (prostaglandin cyclooxygenase), mitochondrial (monoamine oxidase and superoxide dismutase), and soluble enzyme activities (lipooxygenase and superoxide dismutase) were not significantly altered by this treatment. This increase in the enzyme activity with the lipopolysaccharide treatment was abolished with a simultaneous administration of cycloheximide or actinomycin D, and an immunological analysis with antibody for mouse enzyme (rabbit IgG) demonstrated that the observed increment of the enzyme activity was essentially due to an increase in the enzyme protein.     

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.     

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide.

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.     

Antigenicity of filarial superoxide dismutase in human bancroftian filariasis

Superoxide dismutase activity was measured in different stages of growth of filarial parasites (human and cattle). The activity was almost undetected or very low in microfilarial stage but in adult worms, the enzyme activity was high. The enzyme was characterized to be a Cu/Zn superoxide dismutase. Most of the enzyme activity was associated with a detergent extractable fraction of adult (Setaria) parasite. The enzyme was also detected in thein vitro released products of adult worms. The superoxide dismutase activity was completely inhibited with IgG antibody from chronic filarial patients in contrast to IgG from normal people. Filarial patients particularly have high IgG and IgM antibody levels to purified enzyme. However, individuals from non-filarial regions of Orissa are sero-negative for superoxide dismutase antibodies. Antibody response to superoxide dismutase could thus be used for filarial diagnosis.     

Alteration of free radical metabolism in the brain of mice infected with scrapie agent.

Alteration of free radical metabolism in the mouse brain by scrapie infection was evaluated. The infection of mice with scrapie agent, 87V strain, slightly increased the activities of catalase and glutathione-S-transferase, while it had no effect on glutathione peroxidase, glutathione reductase, and Cu, Zn-superoxide dismutase. Results show that the scrapie infection decreased the activity of mitochondrial Mn-superoxide dismutase by 50% but increased that of monoamine oxidase (p < 0.05). Scrapie infection also increased the rate of mitochondrial superoxide generation (p < 0.05). Following scrapie infection, the level of free-sulfhydryl compounds in brain homogenates slightly decreased, but the content of thiobarbituric-acid-reactive substances and malondialdehyde increased significantly. Electron microscopy indicated that the ultrastructure of mitochondria was destroyed in the brain of scrapie-infected mice. These results suggest that elevated oxygen free radical generation and lowered scavenging activity in mitochondria might cause the free radical damage to the brain. Such deleterious changes in mitochondria may contribute to the development of prion disease.