Modulation of class Pi glutathione transferase activity by sulfhydryl group modification.
Glutathione transferases (GSTs) in Class Pi (rat GST-P (7-7) and human GST-pi) were inactivated by treatment with 0.05-1 mM hydrogen peroxide (H2O2), while GSTs in Class Alpha (1-2) and Class Mu (3-3, 3-4) were not, even with 5 mM H2O2. In the presence of 1 mM reduced glutathione (GSH), the inactivated GST-P (-pi) was effectively reactivated by the action of thioltransferase, which had been partially purified from rat liver by GSH-Sepharose affinity chromatography and gel filtration using Sephadex G-75. Thus, inactivation of GST-P by H2O2 was indicated to involve concomitant formation of disulfide bonds between cysteinyl residues. Single GST-P or GST-pi subunits are known to have four cysteinyl residues at the same positions, which can react with sulfhydryl group modifiers. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, GST-P treated with 1 mM H2O2 showed several extra bands, at least three, with apparent molecular weights of 21.5, 18, 37 kDa in addition to the native GST-P subunit band with a molecular weight of 23.5 kDa. These extra bands were identified as inactive forms since they returned to the native band with accompanying restoration of the activity when treated with dithiothreitol, mercaptoethanol, or thioltransferase. Disulfide bonds were formed mainly within subunits, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed.
Shen
H X
HX
Second Department of Biochemistry, Hirosaki University School of Medicine, Japan.
Tamai
K
K
Satoh
K
K
Hatayama
I
I
Tsuchida
S
S
Sato
K
K
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology*; D005260:Female; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000378:metabolism
D007527:Isoenzymes; D013438:Sulfhydryl Compounds; D006861:Hydrogen Peroxide; D005982:Glutathione Transferase; D004229:Dithiothreitol