Inactivation of mouse liver glutathione S-transferase YfYf (Pi class) by ethacrynic acid and 5,5'-dithiobis-(2-nitrobenzoic acid).
Mouse liver glutathione S-transferase YfYf (Pi class) reacts with [14C]ethacrynic acid to form a covalent adduct with a stoichiometry of 1 mol per mol of subunit. Proteolytic digestion of the enzyme-[14C]ethacrynic acid adduct with V8 protease produced an 11 kDa fragment containing radioactivity. Sequencing revealed this to be an N-terminal peptide (minus the first 15 residues, terminating at Glu-112) which contains only one cysteine residue (Cys-47). This is tentatively identified as the site of ethacrynic attachment. Kinetic studies reveal that glutathione S-conjugates protect against inactivation by ethacrynic acid, but the level of protection is not consistent with their potency as product inhibitors. A model is proposed in which glutathione S-conjugates and ethacrynic acid compete for the free enzyme, and a second molecule of ethacrynic acid reacts covalently with the enzyme-ethacrynic acid complex. The native protein contains one thiol reactive with 5,5'-dithiobis-(2-nitrobenzoic acid) at neutral pH. The resultant mixed disulphide, like the ethacrynic acid adduct, is inactive, but treatment with cyanide (which incorporates on a mol for mol basis) restores activity to 35% of that of the native enzyme.
Phillips
M F
MF
Department of Biochemistry, Trinity College, Dublin, Ireland.
Mantle
T J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004976:Ethacrynic Acid / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D013438:Sulfhydryl Compounds / Q000737:chemistry
D007527:Isoenzymes; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; C030190:S-(2,4-dinitrophenyl)glutathione; D004228:Dithionitrobenzoic Acid; D005982:Glutathione Transferase; D005978:Glutathione; D004976:Ethacrynic Acid; C017514:S-methyl glutathione