Cytosolic glutathione peroxidase from liver of pacu (Piaractus mesopotamicus), a hypoxia-tolerant fish of the Pantanal.
Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.
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Department of Biochemistry, UERJ, Av Professor Manuel de Abreu, 444, Rio de Janeiro, RJ, Brazil.
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D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001555:Benzene Derivatives / Q000378:metabolism; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D000860:Hypoxia / Q000378:metabolism*; D007525:Isoelectric Focusing; D008099:Liver / Q000166:cytology* / Q000201:enzymology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines / Q000378:metabolism; D013020:South America; D053833:Wetlands; D000094765:Glutathione Peroxidase GPX1
D000975:Antioxidants; D001555:Benzene Derivatives; D010713:Phosphatidylcholines; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D005978:Glutathione; C007164:cumene hydroperoxide; D000645:Ammonium Sulfate; D000094765:Glutathione Peroxidase GPX1