Superoxide dismutases from the oyster parasite Perkinsus marinus: purification, biochemical characterization, and development of a plate microassay for activity.
We have isolated and biochemically characterized superoxide dismutase (SOD) activity in cell extracts of clonally cultured Perkinsus marinus, a facultative intracellular parasite of the Eastern oyster, Crassostrea virginica. In order to assess the SOD activity throughout the purification, we developed and optimized a 96-well-plate microassay based on the inhibition of pyrogallol oxidation. The assay was also adapted to identify SOD activity type (Cu/Zn-, Mn-, or FeSOD), even in mixtures of more than one type of SOD. All SOD activity detected in the cell extracts was of the FeSOD type. Most of the SOD activity in P. marinus trophozoites resides in a major component of subunit molecular weight 24 kDa. The protein was purified by affinity chromatography on an anti-SOD antibody-Sepharose column. Amino-terminal peptide sequence of the affinity-purified protein corresponds to the predicted product of the PmSOD1 gene and indicates that amino-terminal processing has taken place. The results are discussed in the context of processing of mitochondrially targeted SODs.
Ahmed
Hafiz
H
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore, MD 21202, USA.
Schott
Eric J
EJ
Gauthier
Julie D
JD
Vasta
Gerardo R
GR
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003486:Cyanides / Q000494:pharmacology; D056890:Eukaryota / Q000201:enzymology* / Q000472:pathogenicity; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010125:Ostreidae / Q000469:parasitology*; D011480:Protease Inhibitors / Q000494:pharmacology; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011748:Pyrogallol; D015203:Reproducibility of Results; D013482:Superoxide Dismutase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D037521:Virulence Factors / Q000302:isolation & purification
D003486:Cyanides; D011480:Protease Inhibitors; D015800:Protozoan Proteins; D037521:Virulence Factors; D011748:Pyrogallol; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase