Purification and immunological characterization of superoxide dismutase of the onion maggot,Delia antiqua

Cytosolic superoxide dismutase (SOD) of the onion maggot, Delia antiqua, was purified to apparent homogeneity by ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and gel filtration chromatographies. Native molecular mass was estimated as 32,000 daltons. SDS-PAGE revealed only one subunit of 16,000 daltons, indicating that SOD is a homodimer. Isoelectric focusing revealed 3 charge isomers of pls 5.3, 5.5, and 5.7. The specific activity of purified SOD was 4,250 U/mg protein. A monoclonal antibody (MAb, aSOD2B7) raised against Delia SOD recognized only SOD of the same genus, but another MAb (aSOD1H11) recognized SOD of Drosophila melanogaster as well. © 1995 Wiley-Liss, Inc.     

Characterization of Superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers

Abstract An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae , and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes , and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.     

The reaction of cysteine with ceruloplasmin copper

The kinetics of decay in absorbance at 610 nm in the reaction of cysteine with ceruloplasmin was biphasic under anaerobic conditions. Admission of oxygen to the bleached ceruloplasmin restored the blue color to about 75 % of the original value. However, under aerobic or anaerobic conditions an initial bleaching corresponded to a 25 % decrease in blue color. This change was irreversible and remained after removal of excess cysteine from the reaction mixture by dialysis. There was no correlation between transient and steady-state kinetic parameters. Circular dichroism measurements showed a characteristic reduction in the negative band at 450 nm, which is specific for type 1b copper. Isolation and further studies on cysteine-modified ceruloplasmin with a lower A₆₁₀/A₂₈₀ ratio showed < 10% reduction in enzyme activity toward p-phenylenediamine and o-dianisidine. Evidence is also presented that ceruloplasmin catalyzes the oxidation of cysteine with a one-electron reduction of oxygen and the formation of superoxide ion, which is then converted to H₂O₂ by ceruloplasmin. The effect of superoxide dismutase and catalase also confirms the presence of superoxide and H₂O₂. In sum, these data show that a permanent reduction of type 1b copper occurred when cysteine was used as a substrate. We conclude that there is a single electron transfer from cysteine directly to oxygen using one specific copper of ceruloplasmin, type 1b.     

Superoxide dismutase isoenzymes in bovine and human milk

The presence of superoxide dismutase in bovine and human milk was investigated by ultrafiltration, gel filtration, and isoelectric focusing. Conclusive evidence for the presence of this enzyme in both milks is presented. The molecular weight of the enzyme was estimated by gel filtration on Sephadex G-100 to be 30,000, which is consistent with reported values for the copper, zinc form of superoxide dismutase. In addition, enzyme activity was inhibited by cyanide, thus eliminating the possibility that the enzyme was present in the manganese form. Several isoenzymes were detected by isoelectric focusing in polyacrylamide gel, and the isoenzyme pattern in bovine milk was the same as that found for bovine plasma, suggesting that milk superoxide dismutase originates from plasma. It may be that the presence of copper, zinc superoxide dismutase in milk is important for the maintenance of its oxidative stability.     

血红细胞超氧化物歧化酶(SOD)制备工艺的研究初报

本文报告了本实验室设计的由血红细胞自溶液60℃热变性, 乙醇——氯仿法除血红蛋白,旋转蒸发法减压浓缩抽去氯仿、乙醇,硫酸铵分级盐析法沉降SOD,Sepbadex G-75层析提纯SOD等步骤构成的一条成本低、设计合理、简便实用的分离纯化SOD的工艺路线。     

羊红细胞超氧化物歧化酶的研究

自羊红细胞分离得到一种高等电点的铜.锌-超氧化物歧化酶(Cu.ZnSOD)。其沉降系数(S)为3.23,亚基分子量为16600,等电点为8.50,紫外最大吸收峰位于259nm,酶分子中含有铜和锌,氨基酸组成特点与其它动物来源的Cu.Zn-SOD相同。该酶的比活性为5500U/mg(黄嘌吟氧化酶—细胞色素还原法);对KCN的抑制作用敏感,最适pH值为6。     

Protective Effects of Human Recombinant Mnsod in Adjuvant Arthritis and Bleomycin-Induced Lung Fibrosis

We have previously shown that human recombinant methionyl manganese superoxide dismutase (MnSOD) is more efficient than CuZnSOD as an anti-inflammatory agent in a model of acute inflammation (Carrageenan-induced pau edema). This effect was attributed to the prolonged half-life of MnSOD in blood (t_1/2 = 6 h vs. 10 min. respectively). In the present study, the two enzymes were compared in terms of their effectiveness in two systems: (I) Adjuvant-induced arthritis in rats, which is considered to be a model for the chronic situation of rheumatoid arthritis and (2) Bleomycin-induced lung fibrosis. which is a chronic situation believed to be mediated by oxygen free radicals.

Rats inflicted with adjuvant arthritis were treated during the period of maximal joint swelling (Days 15-21 after adjuvant injection) with MnSOD or CuZnSOD (12 to 50mg/kg, i.p. daily). MnSOD administration resulted in a 50-75% reduction of paw swelling, as well as inhibition of the elevation of serum globulins. A similar treatment with CuZnSOD gave merely marginal effects.

In the second system, lung fibrosis was induced in rats by intratracheal administration of bleomycin. MnSOD (50mg/kg, s.c.), administered 2 h before and then 2 and 4 days after bleomycin, markedly inhibited lung fibrosis, as evident from lung weight and collagen content measured by the 3rd week. By contrast, CuZnSOD administration did not give a significant effect. The results indicate that MnSOD is superior to CuZnSOD in the treatment of chronic inflammatory processes. In addition, they lend further support to the involvement of oxygen free radicals in bleomycin toxicity.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-l-yl and substituted triazol-4-yl derivitives.     

Enhanced Superoxide Production with on Change of the Antioxidant Activity in Gingival Fluid of Patients with Chronic Adult Periodontitis

In the gingival crevicular fluid (GCF) of control and chronic adult periodontitis (CAP) patients there is a spontaneous release of O₂^- radicals from polymorphonuclear leukocytes (PMN). The addition of the exogenous stimuli phorbol myrystate acetate (PMA) decreased the O₂^- formation in control GCF, while in CAP patients produced a marked enhancement of O₂^- generation.

The circulating PMN of control subjects did not show a spontaneous O₂^- formation, differently from CAP patients. On the contrary, a similar O₂^- production was measured when the circulating PMN were stimulated with PMA.

Moreover, the antioxidant activity measured in 10μl of cell free gingival supernatant (GS) of control and CAP patients had the same values by inhibiting 12.6% and 18.9% respectively of the O₂^- formation supported by a xanthine/xanthine oxidase system.

Probably, the protective or destructive effect of PMN in GCF of CAP patients depends on the variations of the rate of O₂^- formation in respect to the intrinsic antioxidant property of GS.     

Cloning of a superoxide dismutase gene from Listeria ivanovii by functional complementation in Escherichia coli and characterization of the gene product.

Summary A gene encoding superoxide dismutase (EC 1.15.1.1., SOD) was isolated from a plasmid library of chromosomal DNA from Listeria ivanovii by functional complementation of an SOD-negative Escherichia coli host. The nucleotide sequence of the cloned gene was determined and contained an open reading frame which codes for a protein of 202 amino acid residues (calculated molecular weight 22 755 Da including the amino-terminal methionine residue). Comparison of the deduced amino acid sequence of L. ivanovii SOD with previously reported SOD amino acid sequences revealed considerable homologies with Fe- and Mn-dependent SODs. Enzymatic analyses using cell lysates and the purified recombinant enzyme indicated that this SOD is manganese-dependent. The recombinant SOD accounted for up to 30% of the total soluble protein in recombinant E. coli and protected sodA sodB mutants against the toxic effects of paraquat. Subunits of the recombinant Listeria SOD and of both E. coli SODS formed enzymatically active hybrids in vivo.Some of our preliminary observations have been published as a conference report of SOD V (Jerusalem, 1989) in Free Rad Res Commun (1991) 12–13:371