The binding of copper ions to copper-free bovine superoxide dismutase. Copper distribution in protein samples recombined with less than stoicheiometric copper ion/protein ratios.
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ABSTRACT:
SUBMITTER: Rigo A
PROVIDER: S-EPMC1164470 | biostudies-other | 1977 Jan
REPOSITORIES: biostudies-other
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Project description:The kinetics of reconstitution of bovine superoxide dismutase from Cu2+ and the copper-free enzyme have been studied by activity, u.v.-absorption, electron-paramagnetic-resonance and pulsed-nuclear-magnetic-resonance measurements. The process appears to be first-order up to 80% completion in most conditions, and is pH-dependent, with an apparent pK of 6.5. U.v.-absorption and solvent proton relaxation rate measurements show that fast binding of Cu2+ occurs, and the initial ligands are likely to be, at least in part, those of the native active site. The recovery of the native activity and spectroscopic properties is a slow process with activation energies of 92 kJ/mol at pH 5.3 and 8.4kJ/mol at pH 8.1 and can be described as a rearrangement of the site around the bound metal. The rate of this process is lower in partially recombined protein samples, probably because of intersubunit interactions.
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Project description:1. E.p.r. (electron-paramagnetic-resonance), proton-relaxation and u.v.-absorption parameters, and enzyme activity of samples of Cu2+-free bovine superoxide dismutase recombined with different amounts of Cu2+ up to the stoicheiometric [Cu2+]/protein] ratio were investigated after attainment of equilibrium in the recovery process. 2. The e.p.r. spectra were identical with the spectrum of the native protein at all [Cu2+]/[protein] ratios. The relaxation rate of the water protons (T1) and the u.v. absorption increase as linear functions of the added Cu2+. 3. On the other hand, in recombination experiments in the range pH 7.6-10.5 the enzyme activity shows a non-linear increase as the [Cu2+]/[protein] ratio rises. The experimental curves can be interpreted in terms of the model of co-operative binding of Cu2+ to the two sites proposed on the basis of the electrophoretic analyses of the samples, and show that the specific activity of the molecules containing only one Cu2+ ion is twice as high as that of the molecules with two Cu2+ ions. 4. These results support the hypothesis of an anti-co-operative interaction between the two sites during the activity, which allows only one Cu2+ ion to function in catalysis.
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Project description:Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed "entry site" for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment.
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Project description:A common and severe neural tube defect (NTD) phenotype, myelomeningocele (MM), results from the defective closure of the caudal end of the neural tube with herniation of the spinal cord and meninges through the vertebral column. The exact mechanisms for NTDs are unknown, but excessive oxidative stress, particularly in association with maternal diabetes, has been postulated as a mechanism for MM.The SNPlex Genotyping (ABI, Foster City, CA) platform was used to investigate single nucleotide polymorphisms (SNPs) across the superoxide dismutase (SOD) 1 and 2 genes to assess their association with MM risk. The study population included 329 trio (affected child and both parents) and 281 duo (affected child and one parent) families. Only cases with documented MM were studied. Seventeen SNPs across the SOD1 and SOD2 genes met the quality-control criteria to be considered for statistical analysis. Genetic association was assessed using the family-based transmission disequilibrium test in PLINK (a genome association analysis toolset).Four SNPs in the SOD1 gene (rs 202446, rs202447, rs4816405, and rs2070424) and one SNP in the SOD2 gene ( rs5746105) [corrected] appeared to be associated with MM risk in our population. After adjusting for multiple testing, these SNPs remained significant.This study provides the first genetic evidence to support association of myelomeningocele with superoxide scavenging. The rare alleles of the five specific SNPs within SOD1 and SOD2 appear to confer a protective effect on the susceptibility for MM risk in the MM population tested. Further evaluation of the roles of superoxide scavenging and neural tube development is warranted.
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Project description:The localization of copper and zinc-superoxide dismutase in normal and neoplastic human skin was examined with immunochemical techniques. Skin samples were taken from males and females of different ages, UV exposed and non-exposed areas and basal-/spino-cellular carcinomas. The enzyme was localized diffusely in the cytoplasm and was also found in the nuclei of epidermal cells, endothelial cells and other dermis cell types. The dismutase content in the epidermis was higher in males than females, UV-exposed than non-exposed and young than old people. In the tumors, the enzyme content of the superficial epidermal layers was higher than in the deep tumoral epithelial cells. These data suggest that the localization of Cu, Zn-SOD in skin tissues reflects the gender and age of the subject, the cell types and their normal or diseased state. Further studies based on the investigation of systemic changes of this enzyme in physiological and pathological epidermis could provide additional information on tumor cell generation.
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Project description:UnlabelledSuperoxide dismutases (SODs) are metalloproteins that protect organisms from toxic reactive oxygen species by catalyzing the conversion of superoxide anion to hydrogen peroxide and molecular oxygen. Chlorovirus PBCV-1 encodes a 187-amino-acid protein that resembles a Cu-Zn SOD with all of the conserved amino acid residues for binding copper and zinc (named cvSOD). cvSOD has an internal Met that results in a 165-amino-acid protein (named tcvSOD). Both cvSOD and tcvSOD recombinant proteins inhibited nitroblue tetrazolium reduction of superoxide anion generated in a xanthine-xanthine oxidase system in solution. tcvSOD was chosen for further characterization because it was easier to produce. Recombinant tcvSOD also inhibited a riboflavin photochemical reduction system in a polyacrylamide gel assay, which was blocked by the Cu-Zn SOD inhibitor cyanide but not by azide, which inhibits Fe and Mn SODs. A k(cat)/K(m) value for cvSOD was determined by stop-flow spectrophotometry as 1.28 × 10(8) M(-1) s(-1), suggesting that cvSOD-catalyzed O2 (-) dismutation was not a diffusion controlled encounter. The cvsod gene was expressed as a late gene, and cvSOD activity was detected in purified virions. Superoxide accumulated rapidly during virus infection, and circumstantial evidence indicates that cvSOD aids its decomposition to benefit virus replication. Cu-Zn SOD homologs have been described to occur in 3 other families of large DNA viruses, poxviruses, baculoviruses, and mimiviruses, which group as a clade. Interestingly, cvSOD does not group in the same clade as the other virus SODs but instead groups in an expanded clade that includes Cu-Zn SODs from many cellular organisms.ImportanceVirus infection often leads to an increase in toxic reactive oxygen species in the host, which can be detrimental to virus replication. Viruses have developed various ways to overcome this barrier. As reported in this article, the chloroviruses often encode and package a functional Cu-Zn superoxide dismutase in the virion that presumably lowers the concentration of reactive oxygen induced early during virus infection.
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