Polymorphism of the glutathione transferase subunit 3 in Sprague-Dawley rats involves a reactive cysteine residue

Comparison of Hirosaki hairless rat (HHR) and Sprague-Dawley (SD) rat liver glutathione transferase (GST) subunits by HPLC revealed differences in sub unit 3; a new peak was detected in HHR GSTs and this was tentatively named X, By chromatofocusing, the HHR CST form composed of peak X and SD rat GST 3-3 were eluted at pH 8.8 and 9.1 respectively. The former was more sensiti ve to the SH reagent N-ethylmaleimide (NEM) than the latter. GSSG treatment of peak X resulted in a shift of retention time (peak Y) by HPLC analysis. However, such conversion was not observed for the SD rat GST 3-3 following GSSG or dithiothreitol (DTT) treatment. Peak Y exhibited m/z values of 260 91.9 and 26125.4 by matrix-assisted laser-desorption ionization-time-of-fli ght MS, higher than those of peak X by 304-307, equivalent to the molecular -mass value of GSH. On treatment with DTT, peak Y was converted into peak X , with release of a substance with HPLC-characteristics of GSH. This substa nce was confirmed to be GSH by liquid chromatography/ MS. These results thu s indicated peak Y to be a glutathionylated form of peak X. Quantification revealed the release of 4 nmol of GSH from 0.12 mg of the peak Y protein, c orresponding to 4.8 nmol (M-r 25 000). The nucleotide sequence of HHR GST s ubunit 3 cDNA proved identical to that reported for pGTA/C44, possessing as paragine and cysteine as the 198th and 199th amino acid residues, respectiv ely, corresponding to lysine and serine in subunit 3 of the SD rat. Thus pe ak X appeared to be the product of HHR GST subunit 3 cDNA. Treatment with N -(4-dimethylamino-3,5-dinitrophenyl)maleimide, a coloured analogue of NEM, followed by trypsin-treatment and sequencing of labelled peptides, identifi ed the reactive cysteine residue of HHR GST subunit 3 to be located at posi tion 199. Unlike SD rat GST 3-3, HHR GST 3-3 was not activated by treatment with xanthine and xanthine oxidase. These results suggest polymorphism of the rat GST subunit 3 gene with individual gene product variation in sensit ivity to oxidative stress.