Characterization of pig liver glutathione S-transferases using HPLC-electrospray-ionization mass spectrometry.
We have characterized 11 porcine liver cytosolic glutathione S-transferase (GST) subunits from their precise molecular mass, immunoreactivity and partial amino acid sequence. Four Alpha-, six Mu- and one unexpected Pi-class GST subunits were found with average molecular masses of 24.984-25.228 kDa, 25.039-25.657 kDa and 23.510 kDa respectively. Molecular masses were established using electrospray-ionization mass spectrometry, with a precision of +/- 3-4 mass units. Glutathione (GSH) and S-hexylglutathione (ShGSH) were tested as affinity ligands in the purification procedure. The binding selectivity of GSH was better than that of ShGSH, although non-GST proteins were retained on both matrices. As already described in other studies, a number of non-GST proteins bound to the affinity resins. Two of them were tentatively identified as mevalonate kinase and carbonyl reductase. The characterization of pig liver cytosolic GST subunits pattern achieved in this work should constitute a useful tool for rapid evaluation of these enzymes' expression in modulation studies.
Rouimi
P
P
Institut National de la Recherche Agronomique, Laboratoire des Xénobiotiques, Toulouse, France.
Anglade
P
P
Debrauwer
L
L
Tulliez
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D005982:Glutathione Transferase / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D017386:Sequence Homology, Amino Acid; D013552:Swine
D007527:Isoenzymes; D005982:Glutathione Transferase