Glutationa S-transferase do cérebro de pacu, Piaractus mesopotamicus (Holmberg, 1887) em diferentes tensões de oxigênio

Abstract

In Pantanal occurs the inundation pulse, a phenomenon that provokes seasonal and circadian oxygen depletion in water of rivers and bacias . Variation of dissolved oxygen alternates the oxygen availability to mitochondria, which results in partial reduction of oxygen in cells. Whether oxygen molecules are not fully reduced to H2O, oxygen ions known as reactive oxygen species (ROS) can escape from the respiratory chain. ROS are strong oxidant, which make them harmful to life in high concentrations. Since pacu, Piaractus mesopotamicus (Holmberg, 1887), is a fish species that has endured large variation of oxygen concentration all along its evolution in Pantanal, we have decided to research to find out whether there are antioxidant enzymatic activities capable of quenching ROS in its organs. Material and methods: In this work GST from brains of pacus subjected to normoxia and hypoxia were purified with MagneGSTTM particle by affinity chromatography. Purified fraction was separated in polyacrylamide gel electrophoresis then the kinetic assays were performed using the electrophilic substrates CDNB and 4-HNE. In order to characterize the proteins presents in the purified fraction a two-dimension electrophoresis was conducted and the showed up spots were cut-off and analyzed in mass-spectrophotometer (MALDI ToF-ToF). Results: SDS-PAGE of the purified fraction from brains of pacus showed two protein bands in normoxia group and three bands in the hypoxic group. The remaining activity of the purified fraction, in both groups was about 60% when compared to the total activity found in the cytosolic fraction. Kinetics studies revealed little differences in Vmax and KM values which suggest the participation of several GST isoenzymes. Furthermore, comparison between normoxia and hypoxic kinetics parameters showed different behavior, so it is plausible that GST pool from the normoxia purified fraction are slightly different from the hypoxic group. The results of two-dimensional electrophoresis showed 14 spots with isoelectric points range from 5.9 to 9.0 in normoxia group while the hypoxic group showed 8 spots with isoelectric point range from 6.5 to 9.47. Only one spot of hypoxia group was not shared with the normoxia group. The primary sequence analysis showed 11 amino acids with 100% homology with GSTP1 from Salmo Salar. Conclusions: The whole results suggest that GST activity found in pacu brain may help them to overcome the oxygen depletion that occur circadian and seasonally. Additionally, the GSTP1 may have a crucial role in their survival.