Efficient Production of Active Human Manganese Superoxide Dismutase in Escherichia Coli

Beck, Yaffa; Bartfeld, Daniel; Yavin, Ziva; Levanon, Avigdor; Gorecki, Marian; Hartman, Jacob R.

doi:10.1038/nbt0888-930

Cited by 41 publications

(27 citation statements)

References 35 publications

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“…However, the N-terminal analysis of the 30 residues was identical with those reported by Barra et al [12]. This indicates that the our isolated human Mn-SOD does not occur in a precursor form [17,321. The Stokes' radius of the native enzyme, obtained as subdata from the HPGCILALLS system, was estimated to be 3.8 nm which corresponds to a molecular mass of 75 kDa for a globular protein.…”

Section: Discussionsupporting

confidence: 72%

Human liver manganese superoxide dismutase

Matsuda

Higashiyama

Kijima

et al. 1990

European Journal of Biochemistry

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Manganese superoxide dismutase (Mn-SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low-angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form.Four of the eight cysteine residues in the tetramer reacted with 5,5'dithiobis(2-nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2-mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs,-reactive peptides isolated by activated thiol-Sepharose covalent chromatography. Incubation of Mn-SOD in 1 O h SDS for 2 or 3 days at 25 "C or 5 min at 100 "C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2-mercaptoethanol heated in SDS produced only the 25-kDa monomer species. Essentially, no thiol groups were detected in the 23-kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.

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Section: Discussionsupporting

confidence: 72%

Human liver manganese superoxide dismutase

Matsuda

Higashiyama

Kijima

et al. 1990

European Journal of Biochemistry

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“…Yields of Mn-SOD and mutant protein were on average 50 mg of protein/50 g of bacterial pellet. Human Mn-SOD and the mutant Y34F human Mn-SOD were purified from E. coli using a combination of heat treatment (60°C) and ion exchange chromatography (DE52 and CM52) according to the procedures of Beck et al (11). The purity of the resulting samples was determined on SDS-polyacrylamide gels that showed one intense band.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Product-inhibited Complex in Catalysis by Human Manganese Superoxide Dismutase

Hearn

Nick

et al. 1999

Journal of Biological Chemistry

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“…Human MnSOD was purified from E. coli using a combination of heat treatment (60°C) and ion exchange chromatography (DE52 and CM52) according to the procedures of Beck et al (1988). The purity of the resulting samples of human MnSOD were determined on SDS-polyacrylamide gels that showed one intense band.…”

Section: Methodsmentioning

confidence: 99%

Catalytic Properties of Human Manganese Superoxide Dismutase

Hsu

Hsieh

et al. 1996

Journal of Biological Chemistry

145

158

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The depletion of superoxide catalyzed by human manganese superoxide dismutase (MnSOD) was observed spectrophotometrically by measuring the absorbance of superoxide at 250 -280 nm following pulse radiolysis and by stopped-flow spectrophotometry. Catalysis showed an initial burst of activity lasting approximately 1 ms followed by the rapid emergence of a greatly inhibited catalysis of zero-order rate. These catalytic properties of human MnSOD are qualitatively similar to those reported for MnSOD from Thermus thermophilus (Bull, C., Niederhoffer, E. C., Yoshida, T., and Fee, J. ) showed a strong dependence on pH that could be described by an ionization of pK a 9.4 ؎ 0.1 with a maximum at low pH.Mitochondria are responsible for an extensive consumption of oxygen in cells, and the mitochondrial respiratory chain subsequently causes a large flux of oxygen radicals, a potential source of damage to protein and especially DNA. It is the mitochondrial superoxide dismutase (SOD) 1 that appears to be responsible for protection of these organelles from oxidative damage by catalyzing the decay of the superoxide radical anion.Three forms of SOD have different catalytic metal ions and different distributions (Beyer et al., 1991;Tainer et al., 1991); it is the manganese SOD that is found in mitochondria and prokaryotes, FeSOD is found in prokaryotes, and Cu,ZnSOD occurs primarily in eukaryotes but has been found in some bacteria. The crystal structure of human MnSOD has been determined at 2.2 Å resolution (Borgstahl et al., 1992). It shows the active sites grouped in pairs across the dimer interfaces, imposing a trigonal bipyramidal geometry on the manganese (Borgstahl et al., 1992). The crystal structures of the MnSOD from Thermus thermophilus at 1.8 Å (Ludwig et al., 1991) and from Bacillus stearothermophilus at 2.4 Å (Parker and Blake, 1988) show strong structural similarity with the human enzyme with a similar geometry about the metal. These structures are also similar to that of the FeSOD (Stoddard et al., 1990) including the same ligands to and geometry about the metal.It is now widely accepted that superoxide dismutases carry out catalysis through a redox process in which the metal cycles between oxidized and reduced states.Initial studies using pulse radiolysis of the MnSOD from B. stearothermophilus determined that its catalysis was complicated by the presence of an inactive form of the enzyme that can interconvert to an active form (McAdam et al., 1977a(McAdam et al., , 1977b. Steady-state constants for catalysis by MnSOD from T. thermophilus were obtained by Bull et al. (1991) from stoppedflow experiments, and the rapid emergence of the inactive form of the enzyme was quantitated. Bull et al. (1991) were able to observe the inactive form spectrophotometrically and suggested that it results from oxidative addition of O 2 . to the Mn(II) form of the enzyme, resulting in a side-on complex of Mn(III) and peroxide, Mn(III)O 2 Ϫ2 . We have overexpressed human MnSOD in Escherichia coli and purified this enzyme to homogenei...

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Efficient Production of Active Human Manganese Superoxide Dismutase in Escherichia Coli

Cited by 41 publications

References 35 publications

Human liver manganese superoxide dismutase

Human liver manganese superoxide dismutase

Characterization of the Product-inhibited Complex in Catalysis by Human Manganese Superoxide Dismutase

Catalytic Properties of Human Manganese Superoxide Dismutase

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