Characterization of enzymatically active Onchocerca volvulus Cu/Zn superoxide dismutase expressed in Escherichia coli.
The Onchocerca volvulus superoxide dismutase was expressed in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. The recombinant O. volvulus superoxide dismutase (rOVSOD) was found in the cytosol of the disrupted bacteria and represented > 10% of the total bacterial protein. The enzyme was purified to homogeneity using DEAE-Sepharose chromatography, followed by phenyl-Sepharose chromatography. The rOVSOD was enzymatically active which was demonstrated by its reactivity with O2.- produced either by the xanthine-xanthine oxidase system or by stimulated eosinophils. The specific activity was determined to be 4668 U mg-1. This activity could be blocked by rabbit antiserum raised against the rOVSOD. The maximal activity was obtained upon supplementation of the bacterial growth media and enzyme buffer with copper and zinc ions. Activity characteristics in the presence of inhibitors was also characteristic of a Cu/Zn superoxide dismutase. The rOVSOD has an apparent subunit molecular mass of 16,000 in SDS-PAGE. The active enzyme behaves as a dimer of 32 kDa as determined by gel filtration.
Bernhard Nocht Institute for Tropical Medicine, Department of Biochemistry, Hamburg, Germany.
K
K
Henkle-Dührsen
C
C
Warnecke
N
N
Brattig
E
E
Liebau
R D
RD
Walter
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004804:Eosinophils / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017181:Onchocerca volvulus / Q000201:enzymology* / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
D011994:Recombinant Proteins; D013481:Superoxides; D013482:Superoxide Dismutase