ABSTRACT
Electrophilic compounds are ubiquitous in the environment and aquatic life is inevitably affected. Glutathione S-transferases (GSTs) are a class of enzymes that facilitate the detoxification of these electrophiles in phase II metabolism. In this study, cytosolic GSTs were isolated and characterized from striped bass liver (Morone saxitilis). Nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to elucidate peptide sequences, and the proteins were found to have homology to rho and alpha by searching against the NCBI non-redundant database (nrDB). Catalytic activities of the cytosolic GSTs towards 1-chloro-2,4-dinitrobenzene (CDNB) were determined to be 141+/-34 and 155+/-65nmol/min/mg for males and females, respectively (both n=3). However, sex differences in classes expressed and activity toward CDNB were not statistically significant (p>0.05).
Subject(s)
Bass/metabolism , Cytosol/enzymology , Glutathione Transferase/metabolism , Amino Acid Sequence , Animals , Gene Expression Regulation , Glutathione Transferase/chemistry , Glutathione Transferase/geneticsABSTRACT
Two cytosolic glutathione S-transferase (GST) classes were isolated and characterized from California halibut (Paralichthys californicus) liver. Nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to elucidate peptide sequences and the proteins were identified as theta and alpha by searching against the NCBI non-redundant database (nrDB). Catalytic activity of the cytosolic GSTs towards 1-chloro-2,4-dinitrobenzene (CDNB) was determined to be 0.23+/-0.003 U/mg cytosolic protein.
Subject(s)
Flounder/metabolism , Glutathione Transferase/metabolism , Animals , Cytosol/enzymology , Dinitrochlorobenzene , Glutathione Transferase/isolation & purification , Hepatocytes/enzymologyABSTRACT
This study evaluated the activity and expression of the glutathione S-transferase (GST) detoxification isoenzymes in juvenile white sturgeon (Acipenser transmontanus) and Chinook salmon (Oncorhynchus tshawytscha) during acclimation from freshwater (2 per thousand) to estuarine (15 per thousand) salinity conditions. In white sturgeon, GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) increased significantly (P = 0.005; n = 5) with elevated salinity, but not for the Chinook salmon (P = 0.174; n = 10). GST activity of both sturgeon and salmon toward ethacrynic acid (ETHA) did not significantly change with elevated salinity (P = 0.516 with n = 3, and P = 0.125 with n = 3, respectively). Expression of the GST classes, and hepatic glutathione (GSH) concentration, as determined by HPLC, also did not significantly change with increased salinity. In conclusion, overall GST activity in white sturgeon, but not Chinook salmon, is stimulated by elevated water salinity, thus electrophilic chemicals such as pesticides may be more effectively detoxified by sturgeon as they undergo seaward migration.
Subject(s)
Fishes/metabolism , Glutathione Transferase/metabolism , Salmon/metabolism , Sodium Chloride/pharmacology , Animals , Dinitrochlorobenzene/toxicity , Ethacrynic Acid/toxicity , Glutathione/metabolism , Liver/enzymologyABSTRACT
Four cytosolic glutathione S-transferase (GST) classes were isolated and characterized from juvenile winter run Chinook salmon (Oncorhynchus tshawytscha) liver. Two techniques were used: (1) gel electrophoresis/immunoblotting against a polyclonal striped bass GST antibody and (2) high-pressure liquid chromatography (HPLC). Nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to elucidate peptide sequences and the proteins were identified as pi, theta, mu and alpha, by searching against the NCBI non-redundant database (nrDB). Catalytic activity of the cytosolic GSTs towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETHA) were determined to be 0.3+/-0.05 U/mg cytosolic protein and 0.06+/-0.02 U/mg cytosolic protein, respectively.
Subject(s)
Cytosol/enzymology , Glutathione Transferase/isolation & purification , Salmon/metabolism , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Chromatography, Liquid , Computational Biology , Dinitrochlorobenzene/metabolism , Ethacrynic Acid/metabolism , Glutathione Transferase/genetics , Immunoblotting , Mass Spectrometry , Molecular Sequence Data , Sequence AlignmentABSTRACT
Glutathione S-transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thus preventing toxicity. This study characterized the cytosolic GST classes of juvenile white sturgeon (Acipenser transmontanus) liver, using two methods of isolation. The first, which employed affinity chromatography, electrophoresis and immunoblotting against a polyclonal striped bass GST antibody, yielded two cytosolic GSTs. The GSTs were identified by nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS), peptide mass mapping and MS/MS sequencing, as well as de novo MS/MS sequencing as GST classes pi and mu using the Mascot search engine and the NCBI non-redundant database (nrDB) for both methods. The molecular masses were determined to be 23,548 +/- 23 and 26,027 +/- 23 Da, respectively, using linear matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The second method of isolation, which used affinity chromatography and high-pressure liquid chromatography (HPLC), yielded pi, mu, and possibly two alpha isoforms by MALDI-TOF-TOF, again searching against the NCBI nrDB. The alpha isoforms were determined to have molecular masses of 25,528 +/- 23 and 25,348 +/- 23 Da by electrospray ionization source (ESI)-MS. Overall, it appears that the HPLC method is more sensitive than immunoblotting with the current antibody. Activity of the cytosolic GSTs was evaluated using the substrate 1-chloro-2,4-dinitrobenzene (CDNB) and found to be 2.4 +/- 0.6 U/mg cytosolic protein, and 0.41 +/- 0.05 U/mg cytosolic protein using ethacrynic acid (ETHA).
