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 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).
Donham
Rachel T
RT
Department of Environmental Toxicology, College of Agricultural and Environmental Sciences, University of California, 1 Shields Avenue, Davis, CA 95616-8588, USA.
Morin
Dexter
D
Jewell
William T
WT
Burns
Stephanie A
SA
Mitchell
Alyson E
AE
Lamé
M W
MW
Segall
H J
HJ
Tjeerdema
Ronald S
RS
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D019295:Computational Biology; D003600:Cytosol / Q000737:chemistry*; D004586:Electrophoresis; D005399:Fishes / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics; D015151:Immunoblotting; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA
D005982:Glutathione Transferase