Copper-zinc superoxide dismutase inhibition

Nalbandyan, 1982 Hodgson and Fridovich, 1975 Asada et al., 1975), whereas azide inhibits the enzymes in the following order iron > manganese > copper/zinc superoxide dismutase (Misra and Fridovich, 1978). Diethyldithiocarbamate is another well-characterized inhibitor of the copper/zinc superoxide dismutase (Heikkila et al., 1977). It forms a complex with the copper and removes the metal from all the protein ligands. The copper-diethyldithiocarbamate complex can be separated without affecting the zinc content of the protein (Cocco et al., 1981). 

The generation of O2 from potassium superoxide was also applied to stop-flow procedures. In this method O2 was dissolved in dimethyl sulfoxide and stabilized in 18-crown-6-polyether. This method is useful for mechanistic studies indeed, McClune and Fee (1976) were able to obtain catalytic rate constants for bovine copper/zinc superoxide dismutase as a function of pH in various buffers. More recently the mechanism of catalysis and of anion inhibition of iron superoxide dismutase from E. coli have been examined by this method using a specially constructed stop-flow spectrophotometer (Bull and Fee, 1985). A limitation of the method is that the pre-equilibrium state cannot be properly investigated because of the time resolution of the stop-flow equipment (== 5 msec). 

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. 

An exception to this rule is galactose oxidase, also known to be a good F relaxation probe, but this enzyme is present only in certain fungi (Marwedel et al., 1975). Since manganese and copper/zinc superoxide dismutase can be discriminated in mixtures containing both enzymes by the selective inhibition of the latter one by cyanide, the relaxation can... 

Copper-zinc superoxide dismutase (CuZn-SOD) inhibits cell-mediated oxidation of LDL, but transgenic mice overexpressing CuZn-SOD might have increased atherosclerotic lesion area (388). The role of copper in atherosclerosis is difficult to predict and the studies are conflicting copper is an intrinsic constituent of SOD and ceruloplasmin and a component of Lysyl oxidase (the enzyme involved in collagen synthesis, a major component of ECM). Also, copper ions catalyze oxidative modification of LDL in vitro and possibly in vivo (reviewed in ref. 389). 

Forman and Fridovich (1973) using an indirect assay whereby O2 was generated either by the action of xanthine oxidase on xanthine or by the mechanical infusion of potassium superoxide in tetrahydrofuran. The generated OJ was allowed to react with ferricytochrome c or with tetra-nitromethane and the product formation was monitored spectroscopically. Details of the two assays are given in Section 11.3. Addition of superoxide dismutase inhibits the formation of products. A rate constant of 2 X 10 M sec was determined for all three enzymes. This value agreed with the rate constant determined by pulse radiolysis for the copper/zinc enzyme (Klug-Roth et al., 1973 Fielden et al., 1974). The mechanism of action of the superoxide dismutases has been investigated by the technique of pulse radiolysis which is described in Section II.2. The bovine erythrocyte copper/zinc enzyme is the most studied form as far as the molecular and catalytic properties are concerned (Rotilio and Fielden,... 

All the inhibitions reported so far indicate that there is no known specific inhibitor for the manganese superoxide dismutase. The enzyme from B. stearothermophilus was found to be inhibited by cacodylate (Thornalley et al., 1982) however, the inhibition could only be observed in the xanthine/xanthine oxidase assay and not in the pulse radiolysis assay. No evidence was obtained that cacodylate could be competing with the enzyme for because little or no inhibition was observed when the copper/zinc rather than the manganese enzyme was assayed in the presence of cacodylate. Further investigations revealed that the inhibitory effect is due to a cacodylate anion radical produced by the interaction of hydroxyl radicals (generated by the xanthine/xanthine oxidase reaction) and cacodylate anions. A radical of pamoic acid (4,4 -methylenebis-(3-... 

Zinc is an essential trace element to human health. It plays an important role in membrane stabilization and in cell protection against oxidative stress because it is p>art of structure of superoxide dismutase, the main enzyme in endogenous control of some types of free oxygen radicals. It also inhibits transition metals, such as copper and iron, from ptroducing reactive types of oxygen (Powell, 2000). This metal is also essential for DNA and RNA polymerase, which has an important effect in hepatic regeneration (Sato et al., 2005). 

Risk assessments are conducted under a far different set of guidelines. One of the first efforts to establish an upper limit for zinc intake was proposed by the Environmental Protection Agency (EPA) in the United States during their development of a reference dose for zinc. The reference dose, or RfD, is the level of substance intake that can be sustained for a lifetime without ill-effect. In setting an RfD the toxicologist tends to take a cautious approach. Copper and iron deficiency are known to be induced by high levels of zinc administration for example, a daily intake of 50 mg/day of supplemental zinc has been reported to produce modest inhibition of a red blood cell enzyme called superoxide dismutase. Whether or not this is a toxic effect remains a matter of conjecture to this day (8, 9). However, toxicologists, in the interest of public health, have tended to interpret this as a potentially adverse effect and 50 mg per day of supplemental zinc intake has become a point of reference for toxicity . 

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