Increased levels of peroxisomal active oxygen-related enzymes in copper-tolerant pea plants

The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes containing Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties of Pisum sativum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme Cu,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these peroxisomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O₂⁻, OH) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L.     

Subcellular distribution of superoxide dismutase in leaves of ureide-producing leguminous plants

The subcellular localization of superoxide dismutase (SOD; EC. 1.15.1.1) was studied in leaves of two ureide-producing leguminous plants ( Phaseolus vulgaris L. cv. Contender and Vigna unguiculata [L.] Walp). In leaves of Vigna and Phaseolus , three superoxide dismutases were found, an Mn-SOD and two Cu, Zn-containing SODs (I and II). Chloroplasts, mitochondria, and peroxisomes were purified by differential and density-gradient centrifugation using either Percoll or sucrose gradients. The yields obtained in intact chloroplasts and peroxisomes from Vigna were considerably higher than those achieved for Phaseolus . Purified chloroplasts only contained the Cu, Zn-SOD II isozyme, but in mitochondria both Mn-SOD and Cu, Zn-SOD I isozymes were present. In purified peroxisomes no SOD activity was detected. The absence of SOD activity in leaf peroxisomes from Vigna contrasts with results reported for the amide-metabolizing legume Pisum sativum L. where the occurrence of Mn-SOD was demonstrated in leaf peroxisomes (del Río et al. 1983. Planta 158: 216–224; Sandalio et al. 1987. Plant Sci. 51: 1–8). This suggests that in leaf peroxisomes from Vigna plants the generation of O₂^- radicals under normal conditions probably does not take place.     

Superoxide dismutase in vesicular arbuscular-mycorrhizal red clover plants

The isoenzymatic pattern of Superoxide dismutase (SOD; EC 1.15.1.1) was studied in the symbiosis of Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe-Trifolium prarense L A Cu.Zn-SOD (M, 40500) was found in spores of G. mosseae . while one Mn-SOD (Mn-SOD I) and two Cu.Zn-SODs (Cu.Zn-SOD 1 and Cu.Zn-SOD II) were present in both roots and leaves of T. pratense . Molecular masses for Cu.Zn-SOD I and Cu.Zn-SOD II were 31000 and 34300. respectively. However, when T. prateme and G. mosseae were associated, mycorrhizal roots showed two new iso-zymes, Mn-SOD II and mycCu.Zn-SOD, which have relative molecular masses of 37 800 and 33 300, respectively. The mycCu.Zn-SOD was found to be specific for this association, whereas Mn-SOD II was also present in nodules of Rhizobium-T. pra-tense . Results suggest that both enzymes are induced in the T. praiense roots in response to invasion by mycorrhizal fungi, perhaps as a result of an increase in the generation of O-₂ radicals in plant roots.     

Intraorganellar distribution of superoxide dismutase in plant peroxisomes (glyoxysomes and leaf peroxisomes)

The intraorganellar distribution of superoxide dismutase (SOD) (EC 1.15.1.1) in two types of plant peroxisomes (glyoxysomes and leaf peroxisomes) was studied by determinations of SOD latency in intact organelles and by solubilization assays with 0.2 molar KCl. Glyoxysomes were purified from watermelon (Citrullus vulgaris Schrad.) cotyledons, and their integrity, calculated on the basis of glyoxysomal marker enzymes, was about 60%. Under the same conditions, the latency of SOD activity determined in glyoxysomes was 40%. The difference between glyoxysomal intactness and SOD latency was very close to the percentage of isozyme Mn-SOD previously determined in glyoxysomes (LM Sandalio, LA Del Río 1987 J Plant Physiol 127: 395-409). In matrix and membrane fractions of glyoxysomes, SOD exhibited a solubilization pattern very similar to catalase, a typical soluble enzyme of glyoxysomes. The analysis of the distribution of individual SOD isozymes in glyoxysomal fractions treated with KCl showed that Cu,Zn-SOD II, the major SOD isozyme in glyoxysomes, was present in the soluble fraction of these organelles, whereas Mn-SOD was bound to the glyoxysomal membrane. These data in conjunction with those of latency of SOD activity in intact glyoxysomes suggest that Mn-SOD is bound to the external side of the membrane of glyoxysomes. On the other hand, in intact leaf peroxisomes where only a Mn-containing SOD is present (LM Sandalio, JM Palma, LA Del Río 1987 Plant Sci 51: 1-8), this isozyme was found in the peroxisomal matrix. The physiological meaning of SOD localization in matrix and membrane fractions of glyoxysomes and the possibility of new roles for plant peroxisomes in cellular metabolism related to activated oxygen species is discussed.     

低温胁迫下嫁接对黄瓜叶片SOD和CAT基因表达与活性变化的影响

研究了低温胁迫下嫁接和自根黄瓜叶片Mn-SOD、Cu/Zn-SOD和CAT mRNA基因表达和酶活性变化及其与抗冷性的关系.结果表明:低温胁迫下,嫁接与自根黄瓜叶片Cu/Zn-SOD、Mn-SOD mRNA基因相对表达量变化分别与其Cu/Zn-SOD、Mn-SOD活性变化相吻合,而CATmRNA相对表达量变化与其CAT活性变化并不一致;嫁接黄瓜叶片Cu/Zn-SOD和Mn-SOD mRNA相对表达量及SOD、Cu/Zn-SOD和Mn-SOD活性均高于自根黄瓜,MDA含量和电解质渗漏率均低于自根黄瓜,嫁接黄瓜较高的SOD基因表达量调控的较高SOD活性是其抗冷性强于自根黄瓜的主要因素;嫁接黄瓜的功能叶CAT mRNA相对表达量略高于自根黄瓜,而幼叶CAT mRNA相对表达量低于后者,但两者CAT活性差异不大,说明低温胁迫对嫁接黄瓜叶片CAT mRNA相对表达量及CAT活性的影响不大.     

Detection of manganese superoxide dismutase mRNA in the theca interna cells of rat ovary during the ovulatory process by in situ hybridization

To determine the possible involvement of reactive oxygen species in ovulation, dynamic aspects of superoxide dismutase (SOD) isozyme were studied in the ovaries of rats by in situ hybridization histochemistry. Previously, mRNA levels of ovarian manganese superoxide dismutase (Mn-SOD) were reported markedly to increase whilst enzymic activity of Mn-SOD decreased during the ovulatory process after treating immature rats with 10 and 5 Units, respectively, of pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Levels of Cu/Zn-SOD activity and Cu/Zn-SOD mRNA were reported to remain unchanged throughout ovulation. This increase in the Mn-SOD mRNA level was shown in the present study by in situ hybridization to be localized to the theca interna cells throughout the PMSG/HCG-induced ovulatory process. The observations suggest that the turnover rate of Mn-SOD but not Cu/Zn-SOD increases specifically in the mitochondria of these cells. SOD has been postulated to play important roles in steroidogenesis. The relationship is discussed between mitochondrial functions in steroid-secreting cells and superoxide radicals and related metabolite(s).     

Dynamic aspects of ovarian superoxide dismutase isozymes during the ovulatory process in the rat.

To investigate the role of superoxide dismutase (SOD) in the ovulatory process, SOD isozymes and their mRNAs were determined in the ovary of 22-day-old rats. After treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), ovarian activity of Mn-SOD decreased markedly while Cu/Zn-SOD remained unchanged. However, the ovarian level of mRNA for Mn-SOD markedly increased after hCG-treatment while that for Cu/Zn-SOD decreased only slightly. Ovulation was inhibited by intravenous injection of a long-acting SOD. These results suggested that superoxide radicals in the ovary might play a critical role in the mechanism for hCG-induced ovulation.     

Diethyldithiocarbamate inhibits in vivo Cu,Zn-superoxide dismutase and perturbs free radical processes in the yeast Saccharomyces cerevisiae cells

Copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese superoxide dismutase (Mn-SOD) in some model experiments in vitro demonstrated antioxidant as well as pro-oxidant properties. In the present study, yeast Saccharomyces cerevisiae lacking Mn-SOD were studied using Cu,Zn-SOD inhibitor N-N'-diethyldithiocarbamate (DDC) as a model system to study the physiological role of the yeast Cu,Zn-SOD. Yeast treatment by DDC caused dose-dependent inhibition of SOD in vivo, with 75% inhibition at 10mM DDC. The inhibition of SOD by DDC resulted in modification of carbonylprotein levels, indicated by a bell-shaped curve. The activity of glutathione reductase, isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase (enzymes associated with antioxidant) increased, demonstrating a compensatory effect in response to SOD inhibition by different concentrations of DDC. A strong positive correlation (R2=0.97) was found between SOD and catalase activities that may be explained by the protective role of SOD for catalase. All observed effects were absent in the isogenic SOD-deficient strain that excluded direct DDC influence. The results are discussed from the point of view that in vivo Cu,Zn-SOD of S. cerevisiae can demonstrate both anti- and pro-oxidant properties.     

氯化钠胁迫下嫁接黄瓜叶片SOD和CAT mRNA基因表达及其活性

研究了NaCl胁迫下嫁接和自根黄瓜叶片Cu/Zn-SOD、Mn-SOD和CAT mRNA的表达与其酶活性变化及其MDA含量和电解质渗漏率变化.结果表明：在NaCl胁迫条件下,嫁接黄瓜叶片Cu/Zn-SOD mRNA、Mn-SOD mRNA和CAT mRNA的相对表达量均高于自根黄瓜,SOD、Cu/Zn-SOD、Mn-SOD和CAT活性也均高于自根黄瓜,说明与自根黄瓜相比,嫁接黄瓜叶片较高的Cu/Zn-SOD mRNA、Mn-SOD mRNA和CAT mRNA相对表达量是其维持较高Cu/Zn-SOD、Mn-SOD和CAT活性的重要原因;随着NaCl胁迫时间的延长,嫁接和自根黄瓜叶片Cu/Zn-SOD- mRNA、Mn-SOD mRNA和CAT mRNA的相对表达量均呈上升趋势,但其酶活性变化并不完全一致,说明还有其他因素参与相关酶活性的调控;嫁接黄瓜叶片MDA含量和电解质渗漏率均低于自根黄瓜,说明嫁接黄瓜具有较高的活性氧清除系统,可以减少活性氧物质的危害,提高其耐盐性.     

Changes in Activities of Antioxidant Enzymes in Sunflower Leaves of Different Ages

Role of superoxide dismutase isozymes and other antioxidant enzymes was studied in relation to leaf age in sunflower (Helianthus annuus L. cv. ACC 1508) at pre-flowering and grain filling stages. Relative water content (RWC) did not change much in leaves of different age and at the two stages. Protein content declined continuously from the youngest to the oldest leaf, while chlorophyll (Chl) and carotenoids (Car) contents increased down to 7^th/9^th leaf and declined in subsequent older leaves. Protein, Chl and Car contents were higher at pre-flowering than at seed filling stage. Superoxide dismutase (SOD), its isozymes, and ascorbate peroxidase (APO) and catalase (CAT) activities were highest in the 9^th leaf and declined in subsequent older leaves. SOD and APO activities were higher at seed filling, except in oldest senescent (13^th, 15^th) leaves. Among SOD isozymes, Cu/Zn-SOD and Mn-SOD activities accounted for most of the total SOD, and only marginal activity was observed for Fe-SOD. Peroxidase activity increased from youngest to the oldest leaf at pre-flowering stage and down to 13^th leaf at seed filling stage. This revised version was published online in July 2006 with corrections to the Cover Date.     

Superoxide dismutases of chestnut leaves, Castanea sativa: Characterization and study of their involvement in natural leaf senescence

Superoxide dismutases (SOD; EC 1.15.1.1) in chestnut ( Castanea sativa Mill., cv. 431) leaves were characterized by native polyacrylamide gel electrophoresis. The three molecular forms of SOD were distinguished from each other by their different sensitivity to cyanide and H₂O₂ Three CuZn-containing SODs were detected (CuZn-SOD I, II. and III), and all the isozymes had a molecular mass of 33 kDa. CuZn-SOD III was the most abundant isozyme. whereas CuZn-SOD II was present in a minor amount. In leaves showing typical symptoms of senescence increases of 2.5-. 7- and 4-fold in the specific activities of CuZn-SODs I, II, and III. respectively, were found. In addition, the pattern of the three isozymes was modified by the age of leaves, a rise in the CuZn-SOD II and a decrease in the CuZn-SOD 1 percentages being found in senescent leaves compared to green leaves. As to other activated oxygen-related enzymes, an increase in the superoxide-generating xanthine oxidase activity and a decline in both catalase and peroxidase activities during natural senescence of chestnut leaves were observed. Results obtained suggest that in natural senescence of chestnut leaves activated oxygen species are involved, and an overproduction of hydrogen peroxide and superoxide radicals probably takes place.     

Changes in Superoxide Dismutase Activity in Liver and Lung of Old Rats

Superoxide dismutase activity was measured in liver and lung from 3 and 24 month-old rats. Both total SOD and Mn-SOD activity decreased significantly in the liver of old rats. Recent results from our laboratory have indicated that during aging, the activity of Cu/Zn-SOD decreases in rat liver and that there is an accumulation of altered protein. It was also shown that the old Cu/Zn-SOD had one histidine fewer than the young one. In the present study, the immunoprecipitation experiments showed that the amount of immunoprecipitable Mn-SOD from liver of old rats was greater than from young ones, but when amino acid residues were measured in purified young and old Mn-SOD from liver, no change was observed. In lung, no significant age-related differences in total SOD, Cu/Zn-SOD and Mn-SOD activity were found, nor was there accumulation of altered protein during aging.     

Changes in Superoxide Dismutase Activity in Liver and Lung of Old Rats

Superoxide dismutase activity was measured in liver and lung from 3 and 24 month-old rats. Both total SOD and Mn-SOD activity decreased significantly in the liver of old rats. Recent results from our laboratory have indicated that during aging, the activity of Cu/Zn-SOD decreases in rat liver and that there is an accumulation of altered protein. It was also shown that the old Cu/Zn-SOD had one histidine fewer than the young one. In the present study, the immunoprecipitation experiments showed that the amount of immunoprecipitable Mn-SOD from liver of old rats was greater than from young ones, but when amino acid residues were measured in purified young and old Mn-SOD from liver, no change was observed. In lung, no significant age-related differences in total SOD, Cu/Zn-SOD and Mn-SOD activity were found, nor was there accumulation of altered protein during aging.     

Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O₂⁻ and H₂O₂ produced under PEG-induced water stress in Trifolium repens.     

Characterization of Wild-Type and Amyotrophic Lateral Sclerosis-Related Mutant Cu,Zn-Superoxide Dismutases Overproduced in Baculovirus-Infected Insect Cells

Abstract: We describe the use of a baculovirus expression system to overproduce human Cu,Zn-superoxide dismutase (SOD). Spodoptera frugiperda (Sf21) insect cells infected with a baculovirus carrying the Cu,Zn-SOD cDNA synthesized a large amount of Cu,Zn-SOD apoprotein in the conventional medium. The SOD activity of the apoprotein, which was initially very low, increased in a dose-dependent manner when Cu²⁺ and Zn²⁺ were added to the medium. Cells grown in media supplemented with Cu²⁺ alone exhibited nearly maximal SOD activity. SOD activity reached 40% of the maximal level within 2 h after addition of Cu²⁺ to postinfected cells cultivated for 3 days in the conventional medium, and the activity gradually increased thereafter. The protein produced by the infected cells was purified by a simple procedure involving two chromatographic steps, DE52 ion exchange and ACA54 gel filtration. Identification of the recombinant Cu,Zn-SOD with the human erythrocyte enzyme was confirmed by immunochemical reactivity to anti-human Cu,Zn-SOD antibody and by partial amino acid sequencing of peptides from purified protein (50 amino acid residues in total). We constructed three mutant enzymes, which have been found in familial amyotrophic lateral sclerosis and are overproduced in Sf21 cells, and purified them. Mutant enzymes Gly⁴¹Asp, His⁴³Arg, and Gly⁸⁵Arg exhibited 47, 66, and 99% of wild-type SOD activity, respectively. The availability of this protein will facilitate investigation of the relationship between the structure and function of the mutant enzymes found in familial amyotrophic lateral sclerosis.     

Protein patterns and superoxide dismutase activity in non-mycorrhizal and arbuscular mycorrhizalPisum sativum L. plants

There are few reports in relation to the role of specific proteins in the mycorrhizal symbiosis. Among the changes in the protein expression as a consequence of the arbuscular mycorrhizal symbiosis, only one case related to changes in superoxide dismutase (SOD; EC 1.15.1.1) activity has been reported in the red clover-Glomus mosseae symbiosis.In this paper, the symbiotic system formed by a leguminous plant,Pisum sativum, and the fungusGlomus mosseae is studied in terms of protein patterns and SOD activity in both mycorrhizal and non-mycorrhizal roots. Our results show that among the differential polypeptides separated by SDS-PAGE, one with a molecular weight of 32.0 kDa, and a protein with an isoelectric point of pI 4.9 appeared strongly expressed in mycorrhizal roots. A partial purification of the related polypeptide could be achieved by DEAE-cellulose chromatography. A higher SOD activity was also detected in mycorrhizal pea roots, although both mycorrhizal and non-mycorrhizal roots showed the same isoenzymatic pattern for SODs: two Mn-SODs (I and II) and two Cu,Zn-SODs (I and II) were detected, Cu,Zn-SOD I being the most abundant isozyme in both types of roots. A similar pattern of SOD isozymes (Mn-SODs I and II, and Cu,Zn-SODs I and II) was also found in nodules of mycorrhizal and non-mycorrhizal pea roots. However, in nodules Mn-SOD II was the main isozyme. The bacterial nature of this isozyme is postulated in this report.Dr. Justo Arines died on the 15th November, 1993 in Dijon (France), while he was attending a molecular biology course on mycorrhizas.