Abstract
A copper-deprived form of the enzyme Cu, Zn superoxide dismutase was identifiedin the liver of rats made copper-deficient by dietary restriction. In homogenates ofsuch livers Cu, Zn superoxide dismutase presents a dis-homogeneous electrophoreticprofile with respect to the native enzyme. When rat liver extracts were treated withexogenous copper an electrophoretic pattern resembling the native one was observed.Enzyme purified by chromatography on DE-52 resin shows two major components, onecorresponding to genuine, native enzyme and another one, eluting at higher ionicstrength. The latter protein (Fraction II) consists of several isoforms which showthe same characteristics of the native superoxide dismutase as far as immunoreactivityand molecular weight are concerned, but with decreased contents of copper and zinc. Itscatalytic constant, referring to copper content, was 15 times lower than that obtainedfor the native enzyme. Moreover, the catalytic power of purified Fraction II was notregained upon incubation with copper. The occurrence of a superoxide dismutase voidof metals confirms the hypothesis that this protein plays a dual physiological role:in metal metabolism and in superoxide anion dismutation.
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Argese E, De Carli B, Orsega E, Rigo A, Rotilio G. 1983 A rotating disk electrode for kinetic studies of superoxide dismutase. Applicability in a wide pH range and continuous monitoring of enzyme inactivation. Anal Biochem 132, 110–11.
Autor, AP. 1982 Biosynthesis of mitochondrial superoxide dismutase inSaccharomyces cerevisiae. Precursor form of mitochondrial superoxide dismutase made in the cytoplasm. J Biol Chem 257, 2713–2718.
Bannister JV, Bannister WH, Rotilio G. 1987 Aspects of the structure, function and application of superoxide dismutase. CRC Crit Rev Biochem 22, 111–18.
Beauchamp CO, Fridovich I. 1971 Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44,276–287.
Carrì MT, Galiazzo F, Ciriolo MR, Rotilio G. 1991 Evidence for co-regulation of Cu, Zn superoxide dismutase and metallothionein gene expression in yeast through transcriptional control by copper via the ACE1 factor. FEBS Lett 2278, 263–266.
Ciriolo MR, Desideri A, Paci M, Rotilio G. 1990 Reconstitution of Cu, Zn-superoxide dismutase by the Cu(I) glutathione complex. J Biol Chem 265, 11030–1103.
Culotta VC, Joh HD, Lin SJ, Slekar KH, Strain J. 1995 A physiological role forSaccharomyces cerevisiaecopper/zinc superoxide dismutase in copper-buffering. J Biol Chem 270, 29991–29997.
Ferreira AMDC, Ciriolo MR, Marcocci L, Rotilio G. 1993 Copper (I) transfer into metallothionein mediated by glutathione. Biochem J 292, 673–676.
Fridovich I. 1995 Superoxide radical and superoxide dismutases. Annu Rev Biochem 64, 97–112.
Galiazzo F, Ciriolo MR, Carrì MT, et al. 1991 Activation and induction by copper of Cu/Zn superoxide dismutase inSaccharomyces cerevisiae. Presence of an inactive proenzyme in anaerobic yeast. Eur J Biochem 196, 545–54.
Harris ED. 1992 Regulation of antioxidant enzymes. FASEB J 6, 2675–2683.
Kumagai Y, Shiniyashiki M, San GF, Shimojo N, Sagai M. 1994 An efficient method for purification of cuprozinc superoxide dismutase from bovine erythrocytes. Experientia 50, 673–676.
Kumamaru T, Riordan JF, Vallee BL. 1982Anal Biochem 126, 214–22.
Laemmli UK. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951 Protein measurement with the Folin-phenol reagent. J Biol Chem 193, 265–275.
Petrovic N, Comi A, Ettinger MJ. 1996 Identification of apo-superoxide dismutase (Cu, Zn) pool in human lymphoblasts. J Biol Chem 271, 28331–28334.
Rigo A, Viglino P, Rotilio G. 1975 Polarographic determination of superoxide dismutase. Anal Biochem 68, 1–8.
Rigo A, Viglino P, Calabrese L, Cocco D, Rotilio G. 1977 The binding of copper ions to copper-free bovine superoxide dismutase. Copper distribution in protein samples recombined with less than stoichiometric copper ion/ protein ratio. Biochem J 161, 27–30.
Rossi L, Ciriolo MR, Marchese E, et al. 1994 Differential decrease of copper content and of copper binding to superoxide dismutase in liver, heart and brain of copperdeficient rats. Biochem Biophys Res Commun 203, 1028–103.
Rotilio G, Calabrese L, Bossa F, et al. 1972 Properties of the apoprotein and role of copper and zinc in protein conformation and enzyme activity of bovine superoxide dismutase. Biochemistry 11, 2182–218.
Steinkuhler C, Sapora O, Carrì MT, et al. 1991 Increase of Cu, Zn superoxide dismutase activity during differentiation of human K562 cells involves activation by copper of a constantly expressed copper-deficient protein. J Biol Chem 266, 24580–24587.
Towbin H, Staehlin T, Gordon J. 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76, 4350–435.
Yano S. 1990 Multiple isoelectric variants of copper, zinc superoxide dismutase from rat liver. Arch Biochem Biophys 279, 60–69.
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Rossi, L., Marchese, E., De Martino, A. et al. Purification of a fully metal-depleted Cu, Zn superoxide dismutase from copper-deficient rat liver. Biometals 10, 257–262 (1997). https://doi.org/10.1023/A:1018364130807
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DOI: https://doi.org/10.1023/A:1018364130807