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 subunit 3; a new peak was detected in HHR GSTs and this was tentatively named X. By chromatofocusing, the HHR GST 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 sensitive 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 26091.9 and 26125.4 by matrix-assisted laser-desorption ionization-time-of-flight 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 substance was confirmed to be GSH by liquid chromatography/MS. These results thus 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, corresponding to 4.8 nmol (M(r) 25000). The nucleotide sequence of HHR GST subunit 3 cDNA proved identical to that reported for pGTA/C44, possessing asparagine and cysteine as the 198th and 199th amino acid residues, respectively, corresponding to lysine and serine in subunit 3 of the SD rat. Thus peak 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, identified the reactive cysteine residue of HHR GST subunit 3 to be located at position 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 sensitivity to oxidative stress.
Kumano
T
T
Second Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
Kimura
J
J
Hayakari
M
M
Yamazaki
T
T
Sawamura
D
D
Tsuchida
S
S
D000818:Animals; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism; D008297:Male; D018384:Oxidative Stress; D011110:Polymorphism, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020539:Sequence Analysis, Protein; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D019820:Xanthine / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000494:pharmacology
D018076:DNA, Complementary; D019820:Xanthine; D006861:Hydrogen Peroxide; D014969:Xanthine Oxidase; D005982:Glutathione Transferase; D014357:Trypsin; D005978:Glutathione; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol