Glutathione peroxidase from the white shrimp Litopenaeus vannamei: characterization and its regulation upon pH and Cd exposure.
ABSTRACT Glutathione peroxidase (GPx) is a key enzyme of cellular detoxification systems that defend cells against reactive oxygen species. Knowledge of the complement of GPx in shrimp is essential to understanding regulation and detoxification mechanisms of environmental stress. In this study, we expressed GPx from white shrimp Litopenaeus vannamei in Escherichia coli, and then characterized the purified recombinant enzyme with respect to the effects of pH, temperature on its catalytic activity. Quantitative real-time PCR and western blot analysis were carried out to investigate the expression patterns of GPx in shrimp hepatopancreas exposed to Cd stress. A statistically significant increase in expression of GPx mRNA and protein was observed in the Cd stress at 24 h. By contrast, western blot showed a significant up-regulation in GPx protein expression at 12 h exposed pH stress (5.6 and 9.3, respectively). These results suggest that L. vannamei GPx expression was modulated by Cd and pH stress and may play an important role in detoxification of xenobiotics and antioxidant defense. We conclude that GPx could be used as biomarkers of Cd and pH stress in aquatic environment for the studied species.
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ABSTRACT: Biomarkers of exposure and effect of pollutants were analyzed in croakers Micropogonias furnieri (Teleostei: Sciaenidae) captured in winter and summer in a polluted and in a non-polluted site at the Patos Lagoon estuary (Southern Brazil). Catalase and glutathione S-transferase activities (exposure biomarkers) and lipid peroxidation (effect biomarker) were analyzed in liver samples. Other two effect biomarkers were also studied: blood cells DNA damage (through comet assay and micronucleus test) and respiratory burst measurements. In a broad view, results point to an important seasonal variation of the biochemical biomarkers analyzed. However, data obtained clearly indicate that croakers collected in winter at the polluted site were subjected to a level of clastogenic agents sufficient to generate irreversible genetic damages (mutations) and impair the fish immune system.Marine Pollution Bulletin 03/2006; 52(2):199-206. · 2.53 Impact Factor
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ABSTRACT: A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.Analytical Biochemistry 06/1976; 72:248-54. · 2.58 Impact Factor
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ABSTRACT: cDNA encoding glutathione peroxidase (GPx) mRNA of the white shrimp Litopenaeus vannamei was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA (RACE) using oligonucleotide primers based on the GPx sequence of Homo sapiens (NM002083), Mus musculus (NM008160), Arabidopsis thaliana (U94495), Bos taurus (NM174770), and Capsicum chinense (AJ973135). The 727-bp cDNA contained an open reading frame (ORF) of 567 bp, a 101-bp 5'-untranslated region, and a 59-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid (aa) sequence (189 aa) was 19.25 kDa long with an estimated pI of 8.39. It contains a putative selenocysteine residue which is encoded by the unusual stop codon, TGA, and forms the active site with residues Glu(75) and Trp(153). Comparison of amino acid sequences showed that white shrimp GPx is more closely related to GPx1 and GPx2 than to GPx3 and GPx4 of various animals. The GPx cDNA was synthesized in haemocytes, gills, the hepatopancreas, intestines, and muscles. The respiratory bursts of shrimp increased significantly after a Vibrio alginolyticus injection in order to kill the pathogen, and then induced increases in the activities of SOD and GPx to protect cells against damage from oxidation. However, GPx activity increased as a result of upregulated expression of GPx mRNA which was induced by the increase in H(2)O(2).Fish & Shellfish Immunology 08/2007; 23(1):34-45. · 2.96 Impact Factor