Characterization of glutathione S-transferases in juvenile white sturgeon

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 π and μ 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 π, μ, and possibly two α isoforms by MALDI-TOF-TOF, again searching against the NCBI nrDB. The α 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).