Copper-zinc superoxide dismutase reduced

Copper is part of several essential enzymes including tyrosinase (melanin production), dopamine beta-hydroxylase (catecholamine production), copper-zinc superoxide dismutase (free radical detoxification), and cytochrome oxidase and ceruloplasmin (iron conversion) (Aaseth and Norseth 1986). All terrestrial animals contain copper as a constituent of cytochrome c oxidase, monophenol oxidase, plasma monoamine oxidase, and copper protein complexes (Schroeder et al. 1966). Excess copper causes a variety of toxic effects, including altered permeability of cellular membranes. The primary target for free cupric ions in the cellular membranes are thiol groups that reduce cupric (Cu+2) to cuprous (Cu+1) upon simultaneous oxidation to disulfides in the membrane. Cuprous ions are reoxidized to Cu+2 in the presence of molecular oxygen molecular oxygen is thereby converted to the toxic superoxide radical O2, which induces lipoperoxidation (Aaseth and Norseth 1986). 

Nishikimi et al., (1972) developed an assay for superoxide dismutase, using phenazine methosulfate which is structurally related to flavin. In this assay phenazine methosulfate is reduced by NADH, and on reoxidation, O2 was generated. The O2 was detected with nitroblue tetrazolium, and maximum inhibition of blue formazan formation by superoxide dismutase was 95% indicating that 5% of the reduction was due to direct interaction between the nitroblue tetrazolium and the reduced phenazine methosulfate. Addition of around 30 ng copper/zinc superoxide dismutase resulted in 50% inhibition of formazan formation. The assay was also used to locate the enzyme on polyacrylamide gels. 

Superoxide dismutase will scavenge the Of formed and will therefore inhibit the reduction of the dianisidine radical by Of. Consequently the dianisidine radical will dismute to yield the divalently oxidized dianisidine. In the presence of superoxide dismutase this reaction is augmented (Fig. 6). The possibility that Of could reduce the final product of dianisidine oxidation and reverse the change in absorbance at 460 nm was tested and was excluded. The assay has been used to determine the rate constant for purified swordfish liver copper/zinc superoxide dismutase (Bannister et al., 1979) and could be applied to crude extracts. The assay was also found applicable to polyacryalmide gels (Misra and Fridovich, 1977c). Gels soaked in riboflavin plus dianisidine, and subsequently illuminated, developed stable brown bands. Peroxidases are also stained by this procedure due to the photochemical production of hydrogen peroxide. However, the development of bands due to peroxidase activity is much slower than the development of bands due to dismutase activity. 

Dismutase, superoxide (Nicotiana plumbaginifolia clone pSOD3 copper-zinc protein moiety reduced) 1623b, 4249... 

Cu—Zn superoxide dismutases (SODs) [87,88] are abundant in eukaryotic cells and may serve to protect cells against the toxic effects of superoxide or deleterious oxy-products derived from 02 . The active site copper and zinc ions are 6.3 A apart and are bridged by a histidine imidazolate. In the oxidized form Cu(II) is roughly pentacoordinate, with four His N s and a water molecule. A highly conserved Arg residue is thought to stabilize Cu(II)-bound anions (e.g., Cu(II)—02 ) a redox reaction releases 02, generating Cu(I), which can reduce more 02 substrate to give peroxide and Cu(II). 

Cadmium-113 nuclear magnetic resonance studies of the cadmium substituted bovine superoxide dismutase were carried out Only a very small chemical-shift difference between the 2 Cd(Il) protein (Cd(II) is bound to the zinc site and the copper site is unoccupied) and the 2 Cd(ll)—2 Cu(I) enzyme (analogous to the reduced form of the native protein) was found. This was interpreted in that the imidazolate bridge is protonated at the Cu site after reduction. 

When rats are administered with bacoside A (10 mg/kg), levels of glutathimie, vitamin C, vitamin E, and vitamin A were reduced. The activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were also assayed. Copper, iron, zinc, and selenium levels in brain and serum ceruloplasntin activity were also measured. Administration of bacoside A improved the antioxidant status and maintained the levels of trace elements [16]. 

Susceptibility factors Renal disease Plasma copper, selenium, and zinc concentrations and antioxidant metalloenzymes, glutathione peroxidase (GPX) and superoxide dismutase (SOD), were studied in 17 patients on maintenance hemodialysis, 14 uremic patients, and 14 healthy subjects [46 ]. Plasma selenium concentrations and erythrocyte GPX were significantly lower in those on hemodialysis, and the two were correlated. There was also a correlation between reduced plasma zinc and erythrocyte SOD activity. 

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