Tissue-specific developmental regulation of superoxide dismutase (SOD-1 and SOD-2) activities in genetic strains of mice

The activity levels of CuZn superoxide dismutase (SOD) (SOD-1) and Mn SOD (SOD-2) in liver, kidney, and lung were assessed in newborn and 3-, 10-, 25-, and 70-week-old females from seven genetic strains (BALB/c, Cs^b, C3H/HeSnJ, C3H/S, C57BL/6J, Swiss-Webster, and 129/ReJ) of mice. Total SOD enzyme activity was high at birth and declined somewhat with old age (70 weeks) in the liver and increased in both kidney and lung from newborn to 25 weeks. The activity level of SOD-1 was found to be highly variable among strains at different ages in liver, with little change associated with aging in the kidney, and showed a strain-specific increase during aging in the lung. In general, SOD-2 activity was lower than SOD-1 activity in liver and lung but levels of the two forms of this enzyme were similar in the kidney. The SOD-2 activity increased with age with little variation among strains in kidney. The increase in this form of the enzyme with age was relatively small and strain specific in lung and highly variable among strains in the liver. The Cs^b genotype (acatalasemic) at age 70 weeks showed an exceptionally high SOD-1 activity associated with an exceptionally low SOD-2 activity in the liver. Changes in enzyme activity with age in different tissues associated with differences in activity level among genotypes (of the type reported here for SOD-1 and SOD-2) may be indicative of a complex system of enzyme regulation. Further studies are needed to explain fully the genetic/molecular mechanism(s) for SOD regulation.This study was financially supported by an NSERC operating grant to S.M.S. N.J.S. was the recipient of a postgraduate scholarship from the NSERC during this period.     

A host-specific toxin of Rhizoctonia solani triggers superoxide dismutase (SOD) activity in rice

Changes in the activity of superoxide dismutase (SOD) in rice in response to treatment with Rhizoctonia solani toxin and/or R. solani elicitor were studied. Treatment of rice leaf sheaths with R. solani-toxin significantly increased the SOD activity within 12?h and the maximum enzyme activity was detected 36?h after treatment at which period a fourfold increase in SOD activity was recorded compared to control plants. Isozyme analysis indicated that five new SOD isozymes (SOD-1, SOD-3, SOD-6, SOD-7 and SOD-8) were induced in rice 1?–?2 days after toxin treatment. In elicitor-treated rice leaf sheaths, SOD-2 increased in activity 1?–?5 days after treatment. Pretreatment of rice leaf sheaths with elicitor suppressed the toxin-induced accumulation of SOD.     

Overexpression of Superoxide Dismutase Protects Plants from Oxidative Stress (Induction of Ascorbate Peroxidase in Superoxide Dismutase-Overexpressing Plants)

Photosynthesis of leaf discs from transgenic tobacco plants (Nicotiana tabacum) that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (SOD+) was protected from oxidative stress caused by exposure to high light intensity and low temperature. Under the same conditions, leaf discs of plants that did not express the pea SOD isoform (SOD-) had substantially lower photosynthetic rates. Young plants of both genotypes were more sensitive to oxidative stress than mature plants, but SOD+ plants retained higher photosynthetic rates than SOD- plants at all developmental stages tested. Not surprisingly, SOD+ plants had approximately 3-fold higher SOD specific activity than SOD- plants. However, SOD+ plants also exhibited a 3- to 4-fold increase in ascorbate peroxidase (APX) specific activity and had a corresponding increase in levels of APX mRNA. Dehydroascorbate reductase and glutathione reductase specific activities were the same in both SOD+ and SOD- plants. These results indicate that transgenic tobacco plants that overexpress pea Cu/Zn SOD II can compensate for the increased levels of SOD with increased expression of the H2O2-scavenging enzyme APX. Therefore, the enhancement of the active oxygen-scavenging system that leads to increased oxidative stress protection in SOD+ plants could result not only from increased SOD levels but from the combined increases in SOD and APX activity.     

Modulation of the expression of superoxide dismutase gene in lung injury by 2-chloroethyl ethyl sulfide, a mustard analog

Mustard gas exposure causes inflammatory lung diseases. Many inflammatory lung diseases are associated with oxidative stress. Reactive oxygen species (ROS) are involved in the maintenance of physiological functions. In tissues, it is therefore essential to maintain a steady-state level of antioxidant activity to allow both for the physiological functions of ROS to proceed and at the same time preventing tissue damage. We have recently reported that mustard gas exposure decreases the overall activity of superoxide dismutase (SOD). In the present study, we investigated the effects of mustard gas on each of the three isozymes: SOD-1 (Cu/Zn), SOD-2 (Mn), and SOD-3 (extracellular). Adult guinea pigs were intratracheally injected single doses of 2-chloroethyl ethyl sulfide (CEES) (2 mg/kg body weight) in ethanol. Control animals were injected with vehicle in the same way. The animals were sacrificed after 7 days, and lungs were removed after perfusion with physiological saline. Lung injury was established by measuring the leakage of iodinated-BSA into lung tissue. Mustard gas exposure caused a significant increase in the activity of SOD-1 (35%). However, the SOD-3 activity which is the predominant type in lung was significantly decreased (62%), whereas no change was observed in SOD-2 activity. Thus the decrease in the total activity of SOD was primarily due to the SOD-3 isozyme. Northern blot analysis indicated 3.5-fold increased expression of SOD-1 in mustard gas exposed lung, but no significant change in the expression of SOD-2 and SOD-3 was observed. Mustard gas exposure did not cause mutation in the coding region of SOD-1 gene while causing modulation in expression levels. The protein levels of SOD-1, SOD-2, and SOD-3 were not altered significantly in the mustard gas exposed lung. Our results indicate that the overall decrease in the activity of SOD by mustard gas exposure is probably mediated by direct inactivation of the SOD-3 gene or the enzyme itself. This decrease in the activity of SOD-3 may be due to the cleavage of active form of the protein to an inactive form. The existence of active and inactive forms of SOD-3 as a result of shifts in Cys-Cys disulfide bonding has been described in human, recently. Studies are underway in our laboratory to investigate whether mustard gas induced inactivation of SOD-3 in lung is similarly mediated by a change in Cys-Cys disulfide bonding.     

Conjugates of superoxide dismutase with the Fc fragment of immunoglobulin G.

We constructed conjugates of superoxide dismutase (SOD) and the Fc fragment of human immunoglobulin G. The lysyl residues of bovine erythrocyte Cu,Zn-SOD were covalently linked with cysteine residues of the Fc fragment using N-succinimidyl 4-(N-maleimido)-butylate as a crosslinking agent. Analysis by gel filtration and SDS-PAGE revealed that the conjugates were composed of one molecule of SOD linked with one molecule of Fc [SOD-(Fc)1] and one SOD molecule linked with several Fc molecule [SOD-(Fc)n]. The resulting SOD-Fc conjugates retained more than 90% of the enzyme activity of SOD. When those conjugates were administered intravenously to mice, the half-lives of SOD activity in the circulation were 29 and 42 h for SOD-(Fc)1 and SOD-(Fc)n, respectively, while free SOD had a half-life of 5 min. Intravenous administration of the conjugates to mice markedly repressed the increase in serum glutamic-oxaloacetic transaminase (GOT) activity induced by paraquat. These results suggest that SOD-Fc conjugates, which have long half-lives, effectively perform dismutation of superoxide radicals and may be useful for preventing tissue injury caused by hazardous oxygen metabolites.     

Iso-superoxide dismutase in <Emphasis Type="Italic">Deinococcus grandis</Emphasis>, a UV resistant bacterium

Deinococcus grandis possesses two types of superoxide dismutase (SOD, E. C. 1.15.1.1.) that show distinct electrophoretic behavior, one that migrates slowly and the other that migrates rapidly (SOD-1 and SOD-2, respectively). In this study, SOD-1 was uniformly and abundantly detected, regardless of growth phase, whereas SOD-2 was not detected during early growth, but was detectable from the exponential growth phase. In addition, a substantial increase in SOD-2 was observed in cells that were treated with potassium superoxide or UV, which suggests that SOD-2 is an inducible protein produced in response to stressful environments. Insensitivity of SOD-1 to both H₂O₂ and cyanide treatment suggests that SOD-1 is MnSOD. However, SOD-2 would be FeSOD, since it lost activity in response to H₂O₂ treatment, but not to cyanide. Localization studies of D. grandis iso-SODs in sucrose-shocked cells suggest that SOD-1 is a membrane-associated enzyme, whereas SOD-2 is a cytosolic enzyme. In conclusion, SOD-1 seems to be an essential constitutive enzyme for viability and SOD-2 appears to be an inducible enzyme that is probably critical for survival upon UV irradiation and oxidative stress.     

Genetic and biochemical characterization of Cu,Zn superoxide dismutase mutants in Saccharomyces cerevisiae

The allele scd 1 is a recessive chromosomal mutation in Saccharomyces cerevisiae that eliminates Cu,Zn superoxide dismutase (SOD-1) activity. SOD-1- strains are unable to grow in 100% O2 in rich medium and are methionine and lysine auxotrophic when grown in air (Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J. (1985) Biochem. Biophys. Res. Commun. 130, 533-539). In this report, scd1 was genetically mapped to the right arm of chromosome X, 11 centimorgans proximal to cdc11. The gene for SOD-1 (SOD1) was physically mapped by Southern blot to restriction fragments containing CDC11. scd1 failed to complement a complete deletion of SOD1. Thus, scd1 maps to the SOD1 locus and is designated sod1-1. The molecular basis for the lack of SOD-1 activity in sodl-1 carrying strains has also been established. The size and amount of SOD-1 mRNA in the mutant were essentially the same as in wild type cells. Western blot analysis showed that the SOD-1 dimer and 16-kilodalton subunit that co-migrated electrophoretically with wild type yeast SOD-1 were abundant in mutant cell extracts. However, two additional SOD-1 immunoreactive polypeptides were detected in these extracts in both denaturing and nondenaturing gels. None of the SOD-1 immunoreactive species in the mutant extracts exhibited superoxide dismutase activity. Transformants of the mutant strain carrying episomal, wild type SOD1 expressed wild type, active SOD-1 protein, indicating that the mutant allele had no discernible effect on the correct synthesis and activation of apoSOD-1. Size exclusion chromatography of soluble cell extracts derived from wild type and SOD1 deletion strains identified a copper binding peak that corresponded to SOD-1. This copper-binding fraction was absent in cell extracts from the sod1-1-containing strain although Western blot analysis of the corresponding chromatographic fractions showed that SOD-1 polypeptide was present in these fractions. Sequence data derived from the cloned genes showed that sod1-1 differed from SOD1 only in the adjacent 5'-noncoding region. The biochemical data indicate that this genetic alteration results in the synthesis of a collection of SOD-1 polypeptides that fail to bind copper and may also fail to completely self-associate. Both phenotypes could be due to the inability of these polypeptides to adopt the native SOD-1 conformation.     

Gender differences in myogenic tone in superoxide dismutase knockout mouse: animal model of oxidative stress

Oxidative stress mediated by prooxidants has been implicated in the pathogenesis of vascular disorders. However, the effect of prooxidants on myogenic regulation of vascular function and the differential influence of gender is not known. SOD, an intracellular enzyme, restricts excess prooxidant levels and may limit vascular dysfunction. We therefore tested the effects of Cu,Zn SOD deficiency on vascular tone in both male and female SOD knockout (SOD-/-) mice. We hypothesized that myogenic tone would be enhanced in SOD-/- mice by excess prooxidants compared with wild-type control mice. Indeed, resistance-sized mesenteric arteries from SOD-/- mice exhibited enhanced myogenic tone compared with control mice. Myogenic tone was lower in female than male control mice. Interestingly, this gender effect was absent in SOD-/- mice, such that myogenic tone of mesenteric arteries from females was equated to that of arteries from males. Furthermore, the pathways that modulate myogenic tone were diverse. In both male and female control mice, inhibition of prostaglandin H synthase (PGHS) and nitric oxide synthase (NOS) pathways enhanced myogenic tone. In female SOD-/- mice, inhibition of PGHS and NOS pathways enhanced myogenic tone to a greater extent compared with control mice. Conversely, in male SOD-/- mice, NOS and PGHS inhibition did not alter tone and only inhibition of gap junctions enhanced myogenic tone. In conclusion, this study revealed enhanced myogenic tone in SOD-/- mice compared with control mice. Furthermore, Cu,Zn SOD deficiency particularly enhanced myogenic tone in female mice such that their vascular tone attained the level of male SOD-/- mice, possibly mediated by prooxidants.     

Selection and analysis of superoxide dismutase mutants of Neurospora.

A survey of 12 genetically distinct, heat-sensitive mutants of Neurospora revealed three (un-1, un-3, and un-17) that are specifically deficient in the superoxide dismutase (SOD) isozymes SOD-2 (mitochondrial), SOD-3 (mitochondrial), SOD-4 (exocellular), respectively. Genetic analysis of the three mutants indicates that the enzyme deficiencies are probably the cause of the heat-sensitive phenotype. The phenotypes of the mutants are (1) no growth at the normally optimal temperature 35 degrees C and comparatively inferior growth at 15-30 degrees C; (2) inferior resistance to the oxidants paraquat or oxygen; (3) female sterility; and (4) inferior conidial viability and longevity. Paraquat or O2 inhibition is alleviated respectively by desferrioxamine-Mn (a SOD mimic) and tocopherol. Diverse antioxidants, including tocopherol, are therapeutic for the heat-sensitive and female-sterile phenotypes, and for inferior growth of wild type at stressfully high temperatures. The results support previous theories that heat stress is a form of oxyradical/oxidant stress and that antioxidant enzymes such as SOD are essential for normal growth, development, and longevity. Since the three genes may encode the three enzymes and are not closely linked to either one another or the family of antioxidant-enzyme regulatory genes Age-1, the latter apparently trans-regulate their expression.     

SOD-1 expression in pig coronary arterioles is increased by exercise training

Coronary arterioles of exercise-trained (EX) pigs have enhanced nitric oxide (NO.)-dependent dilation. Evidence suggests that the biological half-life of NO. depends in part on the management of the superoxide anion. The purpose of this study was to test the hypothesis that expression of cytosolic copper/zinc-dependent superoxide dismutase (SOD)-1 is increased in coronary arterioles as a result of exercise training. Male Yucatan pigs either remained sedentary (SED, n = 4) or were EX (n = 4) on a motorized treadmill for 16-20 wk. Individual coronary arterioles ( approximately 100-microm unpressurized internal diameter) were dissected and frozen. Coronary arteriole SOD-1 protein (via immunoblots) increased as a result of exercise training (2.16 +/- 0.35 times SED levels) as did SOD-1 enzyme activity (measured via inhibition of pyrogallol autooxidation; approximately 75% increase vs. SED). In addition, SOD-1 mRNA levels (measured via RT-PCR) were higher in EX arterioles (1.68 +/- 0.16 times the SED levels). There were no effects of exercise training on the levels of SOD-2 (mitochondrial), catalase, or p67(phox) proteins. Thus chronic aerobic exercise training selectively increases the levels of SOD-1 mRNA, protein, and enzymatic activity in porcine coronary arterioles. Increased SOD-1 could contribute to the enhanced NO.-dependent dilation previously observed in EX porcine coronary arterioles by improving management of superoxide in the vascular cell environment, thus prolonging the biological half-life of NO.     

Effect of the free radical-generating herbicide paraquat on the expression of the superoxide dismutase (Sod) genes in maize

10-day-old maize leaves were treated with the oxygen free radical-generating herbicide paraquat for 12 h. Paraquat treatments (10(-5) M) resulted in a 40% increase in superoxide dismutase activity and a smaller increase in catalase activity. The increase in total superoxide dismutase (SOD) activity correlates with higher levels of specific isozymes. The chloroplast (SOD-1) and cytosolic (SOD-2 and SOD-4) forms were increased significantly; however, the mitochondrial form (SOD-3) was increased only slightly. Higher levels of SOD-4 and SOD-3 after paraquat exposure were the result of increased synthesis of these proteins, as determined by labeling in vivo with [35S]methionine. Isolation and in vitro translation of polysomes from 10(-5) M paraquat-treated leaves indicated that paraquat increased the amount of polysomal mRNA which codes for SOD-4 and SOD-3. Superoxide dismutase induction does not appear to be a response that is specific to paraquat, since another superoxide-generating compound, juglone, caused a similar increase in total superoxide dismutase activity. Therefore, the effect of these compounds on the expression of the maize Sod genes is exerted via their ability to generate superoxide.     

有机溶剂可溶的超氧化物歧化酶的制备及其性质

本文报道用谷氨酸、十二醇、葡萄糖酸内酯合成了一种精脂（２Ｃ（１２）ＧＥ）,并制备了ＳＯＤ－糖脂复合体．所得的ＳＯＤ－糖脂复合体是脂溶性的而不是水溶性的,它在乙醇等有机溶剂中的活性比在水中高,而且存在一最适有机溶剂浓度。其对温度、ｐＨ、蛋白酶水解的稳定性比天然ＳＯＤ明显增强。     

大豆黄酮对衰老小鼠脑组织SOD、LDH同工酶的影响

利用SOD和LDH同工酶电泳分析,研究大豆黄酮对衰老小鼠的抗氧化作用。结果显示大豆黄酮没有改变SOD和LDH同工酶谱的特征,但对因衰老引起的小鼠脑组织LDH和SOD同工酶活性、各组分的相对活性和比活力的变化有不同程度的改善作用,即LDH同工酶中LDH-2、LDH-3的活性明显下降,LDH-1的活性下降最为明显,而LDH-4的活性有所下降,但不显著,LDH-5的活性几乎没有变化,SOD同工酶的SOD-1和SOD-2的活性有不同程度的升高。这表明大豆黄酮是通过抑制LDH同工酶H亚基的合成来降低LDH的活性,而对M亚基的合成没有影响,并且能够促进SOD同工酶SOD-1和SOD-2的合成,不影响其遗传稳定性。     

Normal superoxide dismutase-1 (SOD-1) activity with deletion of chromosome band 21q21 supports localization of SOD-1 locus to 21q22

Summary The gene for superoxide dismutase-1 (SOD-1) is clearly on chromosome 21, although there is disagreement on the precise band location of SOD-1 on the long (q) arm of number 21. We report a patient with normal superoxide dismutase-1 (SOD-1) activity and an interstitial deletion of chromosome 21 resulting in monosomy for band q21. His phenotype is characterized by moderate mental retardation, a long narrow face, high and arched palate, cardiac murmur, undescended testes, and long hyperflexible extremities. The normal SOD-1 activity supports localization of this enzyme to 21q22.1.     

Synthesis of Isozymes of Superoxide Dismutase in Maize Leaves in Response to O3, SO2 and Elevated O2

Matters, G. L. and Scandalios, J. G. 1987. Synthesis of isozymesof superoxide dismutase in maize leaves in response to O₃ SO₂and elevated O₂.—J. exp. Bot 38: 842–852. The activities of the enzymes superoxide dismutase (SOD) andcatalase were determined in maize leaves treated with O₃or SO₂for8 h, or with elevated levels of oxygen for up to 96 h. NeitherO₃nor SO₂significantly increased the levels of superoxide dismutaseor catalase activity. However, after 72 h in an atmosphere containing90% oxygen, superoxide dismutase activity was increased, butnot the activities of catalase, ascorbate pcroxidase, and malatedehydrogenase. Immunological analysis showed that amounts ofthe cytosolic superoxide dismutase isozymes, SOD-2 and SOD-4,were increased by the elevated oxygen but not the chroloplast(SOD-1) or mitochondrial (SOD-3) isozymes. Immunoprecipitationof translation products of leaf polysomes indicated that thehigher levels of SOD-2 and SOD-4 were due to increased amountsof polysome-bound mRNA coding for these proteins. The specificresponse of SOD-2 and SOD-4 to 90% oxygen treatments contrastswith the increase in all SOD isozymes in maize leaves treatedwith the herbicide paraquat. Key words: Air pollutants, maize, oxidative stress, oxygen, superoxide dismutase     

Expression of genetically distinct superoxide dismutases in the maize seedling during development

Immunoassays for the cytosolic and mitochondrial superoxide dismutases (SOD) of maize were developed and used to study the expression of these proteins in the maize seedling. The genetically distinct proteins, SOD-3 and SOD-4, are preferentially expressed in the scutellum, comprising approximately 1% of the total water-soluble protein of that tissue. SOD-2, SOD-3, and SOD-4 are synthesized in the scutellum during early sporophytic development, probably on cytosolic ribosomes. Two-dimensional gel electrophoresis of crude scutellar extracts indicates that significant changes occur in the protein composition of the maize scutellum following seed imbibition. Using the immunoassays, a maize line exhibiting a significant reduction in cyanide-sensitive SOD protein was identified.     

Exercise training regulates SOD-1 and oxidative stress in porcine aortic endothelium

Vascular oxidative stress contributes to endothelial dysfunction. Aerobic exercise training improves vascular function. The purpose of this study was to test the hypothesis that exercise training would improve the balance of antioxidant to prooxidant enzymes and reduce markers of oxidative stress in aortic endothelial cells (AEC). Female Yucatan miniature pigs either remained sedentary (SED) or were exercise trained (EX) for 16-19 wk. EX pigs had increased AEC SOD-1 protein levels and Cu/Zn SOD activity of the whole aorta compared with SED pigs. Protein levels of other antioxidant enzymes (SOD-2, catalase) were not affected by exercise training. Protein levels of p67(phox), a subunit of the prooxidant enzyme NAD(P)H oxidase, were reduced in EX vs. SED AEC. These EX adaptations were associated with lower AEC malondialdehyde levels and decreased phosphorylation of ERK-1/2. Endothelial nitric oxide synthase protein, protein nitrotyrosine content, and heme oxygenase-1 protein were not different in EX vs. SED pigs. We conclude that chronic aerobic exercise training influenced both antioxidant and prooxidant enzymes and decreased indexes of oxidative stress in AEC. These adaptations may contribute to improved endothelial function with exercise training.     

Altered levels of antioxidant enzymes associated with two mutations in tomato

Activity of peroxidase, superoxide dismutase and catalase were examined in leaves, stems and roots of olivacea ( oli ) and monstrosa ( mon ) mutants of Lycopersicon esculentum Mill. The extent of the difference between the pattern of oxidative enzyme activities of the wild type (wt) and the mutants was determined. The high peroxidase activity during the developmental stages of the leaves and stems of oli and mon phenotypes is associated with high levels of 4 anodic peroxidases in leaves and of two isozymes in the stem. Leaves of oli exhibit higher activity of the cathodic peroxidase C2, while both mutations have a marked increase of peroxidase C1 in stems. A positive relation between high peroxidase activity and oxidative stress damage was found: in chilling experiments at 5°C, peroxidase level in mutants and wt leaves was negatively correlated with electrolyte leakage. Superoxide dismutase (SOD) activity rises in oli stems around flowering time due to the high activity of the chloroplast forms SOD-1 and SOD-2. Catalases (CAT) were detectable only in early stages of plant development; CAT-2 was nearly absent in wild type tissues but well represented in mon and oli. The oli and mon mutations may affect critical steps of a regulatory pathway controlling various classes of oxidative enzymes in tomato.     

Dosage effects for superoxide dismutase-1 in nucleated cells aneuploid for chromosome 21.

Assays of the activity of chromosome 21 determined superoxide dismutase-1 (SOD-1) in lymphocytes and polymorphonuclear granulocytes have demonstrated 38% and 40% increases, respectively, in cells from individuals with trisomy 21. Similarly, SOD-1 activity in trisomic fibroblasts is increased by 81%, while cells monosomic for chromosome 21 have only 60% of normal activity. Taken together with the data on SOD-1 activities in trisomic erythrocytes and platelets, the present results firmly confirm the existence of a true dosage effect for this enzyme in cells aneuploid for chromosome 21. However, the results of assays of the activity of glutathione peroxidase in trisomic fibroblasts did not confirm the possibility previously reported of a chromosome 21 related dosage effect for this enzyme.