The amino-acid sequence of rat Cu-Zn superoxide dismutase

The primary structure of Cu-Zn superoxide dismutase isolated from rat liver was determined. The enzyme was reduced, carboxymethylated and fragmented by treatment with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by gel filtration or high performance liquid chromatography and sequenced by automated Edman degradation. The total sequence of 153 amino-acid residues per subunit was reconstructed from overlapping peptides. Rat Cu-Zn superoxide dismutase proved to be closely related to the corresponding sequences of other mammals in having more than 80% identical amino-acid residues in homologous position and an acetylated N-terminus. Comparison of the rat Cu-Zn superoxide dismutase structure with those of other species suggests a similar phylogenetic distance between rat, man, pig, cattle and horse and a rapid molecular divergence during vertebrate development compared to earlier evolutionary periods.     

The amino-acid sequence of rabbit Cu-Zn superoxide dismutase

The primary structure of Cu-Zn superoxide dismutase from rabbit liver was investigated. The reduced and S-carboxymethylated enzyme was treated with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by high-performance liquid chromatography and sequenced by automated Edman degradation. With the exception of the N- and C-terminus the complete sequence was established by means of overlapping peptides. The N-terminus is blocked and thus not susceptible to Edman degradation. The amino-acid composition of the tryptic N-terminal peptide corresponds to that of the cytoplasmatic Cu-Zn superoxide dismutases of other mammals investigated. The chromatographic behaviour of these N-terminal peptides on a reversed phase C18 column is also identical, thus suggesting also for the rabbit Cu-Zn superoxide dismutase the N-terminal sequence Ac-Ala-Thr-Lys. The C-terminus was demonstrated to have the sequence -Ile-Ala-Pro by enzymatic degradation with carboxypeptidase Y. The complete amino-acid sequence of the rabbit Cu-Zn superoxide dismutase consists of 152 amino-acids and shows the expected homology to other Cu-Zn enzymes published so far. The aspartate and six histidine residues known to complex the metal ions are conserved at homologous positions. This also applies for the arginine residue near the C-terminus which is supposed to direct the anionic superoxide radical towards the active centre of the enzyme. The amino acid sequence of the rabbit Cu-Zn superoxide dismutase corresponds to those of other mammals in more than 80% of its amino-acid residues. From a total of 152 amino-acid residues the rabbit shares with rat 128, with mouse 130, with horse 127, with pig 126/127, with cattle 130 and with man 131 amino acids in homologous positions. However the Cu-Zn superoxide dismutases of closely related mammals like rats and mice differ in only five amino acid residues of their sequence. A phylogenetic closer relatedness between lagomorphs and rodents than between other orders of mammals, could not be derived from the sequence data given. Rather rodents and lagomorphs are to be considered as two evolutionary independent orders of mammals.     

The primary structure of porcine Cu-Zn superoxide dismutase. Evidence for allotypes of superoxide dismutase in pigs

Cu-Zn superoxide dismutase was purified to homogeneity from mixed pig blood and from a single pig. The isolated product had an absorption ratio 280/260 nm of 0.91, a specific activity of 3 000 +/- 200 units (cytochrome c reduction test), and an isoelectric point of 7.5 (chromatofocusing) or 7.25 (isoelectric focusing), respectively. Sequence determination was performed by automated solid-phase Edman degradation of fragments of the reduced S-carboxymethylated proteins obtained by digestion with trypsin or Staphylococcus aureus proteinase V8 or treatment with cyanogen bromide. Acetylation of the N-terminus was confirmed by comparing high performance liquid chromatography retention times of N-terminal peptides with those of authentic samples. Sequencing of the superoxide dismutase of mixed porcine blood revealed heterogeneity (70% Leu; 30% Val at position 29), whereas the sample derived from a single French pig proved to be homogeneous (100% Leu at position 29). The complete sequence of pig superoxide dismutase comprised 152 amino-acid residues, which corresponds to a theoretical molecular mass of 15 800 Da per subunit, and exhibited the expected high homology with those of other mammals. The aspartate and all 7 histidine residues known to complex the metal ions in bovine superoxide dismutase are conserved in the porcine sequence at the homologous positions Asp82 and His45, His47, His62, His70, His79, His119, respectively.     

Deletions encompassing the manganese superoxide dismutase gene in the Drosophila melanogaster genome.

Two deletions, Df(2R)Sod2-11 and Df(2R)Sod2-332, are recovered that encompass the manganese superoxide dismutase (MnSOD) gene or a null mutant referred to as SOD2n283 in Drosophila. Molecular analysis has revealed that the Df(2R)Sod2-332 deletion completely uncovered both MnSOD and its adjacent gene, Arp53D, whereas Df(2R)Sod2-11 was missing the promoter region of MnSOD gene. As a consequence of reduced MnSOD expression, these deletion heterozygotes are now sensitive to oxidative stress. Complementation analysis with some recently recovered deletions in the 53C/D region has established that other essential loci exist in this interval, and second, that Arp53D function is not essential for the survival of the organism. These deletions will be instrumental in the recovery of missense substitutions in the MnSOD peptide and their influence on oxidative stress resistance.     

Complete amino acid sequence of copper-zinc superoxide dismutase from Drosophila melanogaster

The complete amino acid sequence of the Drosophila melanogaster Cu,Zn superoxide dismutase subunit has been determined by automated Edman degradation. Sequence analyses were performed on the intact S-carboxymethylated protein, two fragments derived from CNBr cleavage, and three peptides recovered from mouse submaxillary protease digestion of the reduced and S-carboxymethylated enzyme. The peptides were aligned by characterizing peptides yielded by trypsin and Staphylococcus aureus V8 protease. All the peptides studied were purified exclusively by reverse-phase columns of HPLC and were analyzed with an improved liquid-phase sequencer. A molecular weight of 15,750 (subunit) was calculated from the 151 residues sequenced. The amino acid sequence of the Drosophila superoxide dismutase subunit is compared with that of four other eucaryotes: man, horse, cow, and yeast. Comparison of the five primary structures reveals very different rates of evolution at different times. Copper-zinc superoxide dismutase appears to be a very erratic evolutionary clock. Val-Val-Lys-Ala- Val-Cys-Val-Ile-Asn-Gly-Asp-Ala-Lys-Gly-Thr-Val-Phe-Phe-Glu-Gln- Glu-Ser-Ser-Gly-Thr-Pro-Val-Lys-Val-Ser-Gly-Glu-Val-Cys-Gly-Leu- Ala-Lys-Gly-Leu-His-Gly-Phe-His-Val-His-Glu-Phe-Gly-Asp-Asn-Thr- Asn-Gly-Cys-Met-Ser-Ser-Gly-Pro-His-Phe-Asn-Pro-Tyr-Gly-Lys-Glu- His-Gly-Ala-Pro-Val-Asp-Glu-Asn-Arg-His-Leu-Gly-Asp-Leu-Gly-Asn- Ile-Glu-Ala-Thr-Gly-Asp-Cys-Pro-Thr-Lys-Val-Asn-Ile-Thr-Asp-Ser- Lys-Ile-Thr-Leu-Phe-Gly-Ala-Asp-Ser-Ile-Ile-Gly-Arg-Thr-Val-Val-Val- His-Ala-Asp-Ala-Asp-Asp-Leu-Gly-Gln-Gly-Gly-His-Glu-Leu-Ser-Lys- Ser-Thr-Gly-Asn-Ala-Gly-Ala-Arg-Ile-Gly-Cys-Gly-Val-Ile-Gly-Ile- Ala-Lys.     

Inhibition of yeast ribosomal stalk phosphorylation by Cu-Zn superoxide dismutase

Reversible phosphorylation of acidic ribosomal proteins of Saccharomyces cerevisiae is an important mechanism, regulating the number of active ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. A new protein-inhibitor regulating activity of PK60S kinase has been purified from yeast extracts and characterised. Peptide mass fingerprinting (PMF) and amino-acid sequence analysis by Post Source Decay (PSD) have identified the inhibitor as a Cu-Zn superoxide dismutase (SOD). Inhibition by SOD is competitive with respect to protein substrates-P proteins and 80S ribosome-with K(i) values of 3.7 microM for P2A protein and 0.6 microM for 80S ribosomes. A close correlation was found between the state of phosphorylation of P proteins in diauxic shift and logarithmic growth yeast cells and activity of SOD. The possible mechanism of regulation of PK60S activity, and participation of SOD protein in regulation of 80S-ribosome activity in stress conditions, is discussed.     

叶绿体超氧化物歧化酶（ChlSOD）基因的构建及序列分析

构建叶绿体超氧化物歧化酶基因（ChlSOD),采用RT-PCR方法分离豌豆RUBP羧化酶小亚基导肽基因（TP）,定向克隆至pUC19测序,定向克隆烟草MnSOD成熟蛋白基因（SODm)至pUC19;采用平粘端连接法将二者在pUC19中构成嵌合基因ChlSOD,并对此基因进行序列分析,序列分析表明：TPcDNATP,12bp的Linker及615bp SODm。TP与ChlSOD基因的序列分析与国外报道序列完全吻合。     

Demonstration of Cu-Zn superoxide dismutase in rat liver peroxisomes. Biochemical and immunochemical evidence.

In this study, by using highly purified rat liver peroxisomes, we provide evidence from analytical cell fractionation, Western blot, and immunocytochemical analysis that Cu-Zn superoxide dismutase is present in animal peroxisomes. Treatment with ciprofibrate, a peroxisome proliferator, increased the peroxisomal superoxide dismutase activity by 3-fold with no effect on mitochondrial activity but a marked decrease in cytosolic superoxide dismutase activity, further supporting that besides cytosolic and mitochondrial localization, Cu-Zn superoxide dismutase is present in peroxisomes also. Demonstration of superoxide dismutase in peroxisomes suggests a new role for this organelle in pathophysiological conditions, such as ischemia-reperfusion injury.     

Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants.

A gene (sod) encoding superoxide dismutase (SOD) was cloned from Streptococcus mutans in Escherichia coli, and its nucleotide sequence was determined. The presumptive amino acid sequence of its product revealed that the SOD is basically of Mn type. Insertional inactivation of the sod gene resulted in the loss of SOD activity in crude extracts, indicating that the gene represents the only functional gene for SOD in S. mutans. Moreover, Southern blot analysis indicated that the S. mutans chromosome had no additional gene which was hybridizable with an oligonucleotide probe specific for an SOD motif. The SOD-deficient mutants were able to grow aerobically, albeit more slowly than the parent strains.     

Interleukin 1 slowly increases lung fibroblast Cu-Zn superoxide dismutase activity levels

Certain pulmonary stress raises rat lung Cu-Zn superoxide dismutase (SOD) activity levels, but cytokines released during stress are reported to exert no regulatory effects on Cu-Zn SOD levels in cultured cells. In contrast, our study found that interleukin 1 (IL-1) can increase Cu-Zn SOD activities in human W138 lung fibroblasts. The difference in results could be explained by differences in experimental conditions. The increases seen here did not occur during the first 24 hr, but Cu-Zn SOD activities more than doubled by 3 days. In addition, little increase occurred unless the medium was changed at 24-hr intervals. On the other hand, some other potential experimental variables showed little or no effects on IL-1-induced increases in Cu-Zn SOD activities. These variables included IL-1 isoform (alpha, beta, or both), IL-1 concentration (0.5, 2, 5, or 7 units IL-1 alpha/ml medium), and the presence or absence of exogenously added copper as CuO or ceruloplasmin. In addition, combining IL-1 with dexamethasone, a synthetic glucocorticoid that enhances some IL-1 actions, produced only additive, not synergistic, increases in Cu-Zn SOD activities. In conclusion, IL-1, in several different experimental protocols, raised lung fibroblast Cu-Zn SOD activity levels, but only after a 1 day lag time. Stress-induced increases in Cu-Zn SOD activity levels in vivo also tend to occur only after lag times.     

Overexpression of Mn-containing superoxide dismutase in transgenic Drosophila melanogaster.

The general objective of this study was to examine the role of mitochondria in the aging process. Two alternative hypotheses were tested: (i) that overexpression of Mn superoxide dismutase (Mn SOD) in the mitochondria of Drosophila melanogaster would slow the accrual of oxidative damage and prolong survival or (ii) that there is an evolved optimum level of superoxide anion radical, such that overexpression of Mn SOD would have deleterious or neutral effects. Microinjection and mobilization of a transgene, which contained a 9-kb genomic sequence encoding Mn SOD, produced 15 experimental lines overexpressing Mn SOD by 5-116% relative to the parental y w strain. Comparisons between these lines and control lines containing inserted vector sequences alone indicated that the mean longevity of the experimental lines was decreased by 4-5% relative to controls. There were no compensatory changes in the metabolic rate, level of physical activity, or the levels of other antioxidants, namely Cu-Zn SOD, catalase, and glutathione. There were no differences between groups in rates of mitochondrial hydrogen peroxide release, protein oxidative damage, or resistance to 100% oxygen or starvation conditions. The experimental lines had a marginally increased resistance to moderate heat stress. These results are consistent with the existence of an optimum level of Mn SOD activity which minimizes oxidative stress. The naturally evolved level of Mn SOD activity in Drosophila appears to be near the optimum required under normal conditions, although the optimum may be shifted to a higher level under more stressful conditions.