S-(4-Bromo-2,3-dioxobutyl)glutathione: a new affinity label for the 4-4 isoenzyme of rat liver glutathione S-transferase.
S-(4-Bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, has been synthesized and characterized by UV spectroscopy and thin-layer chromatography, as well as by bromide and primary amine analysis. Incubation of S-BDB-G (200 microM) with the 4-4 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. The kobs exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 1000 microM, with a kmax of 0.078 min-1 and K1 = 66 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, completely protects against inactivation by S-BDB-G. About 1.3 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated concomitant with 100% inactivation, whereas only 0.48 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when activity is fully retained. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with NaBH4, carboxymethylated, and digested with trypsin. The tryptic digest was fractionated by reverse-phase high-performance liquid chromatography. Two radioactive peptides were identified: Lys82-His-Asn-Leu-X-Gly-Glu-Thr-Glu-Glu-Glu-Arg93, in which X is modified Cys86, and Leu109-Gln-Leu-Ala-Met-CmCys-Y-Ser-Pro-Asp-Phe-Glu-Arg121 , in which Y is modified Tyr115. Only the Lys82-Arg93 peptide was modified in the presence of S-hexylglutathione when the enzyme retained full activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Katusz
R M
RM
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000345:Affinity Labels / Q000138:chemical synthesis*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005978:Glutathione / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D000345:Affinity Labels; D007527:Isoenzymes; D010446:Peptide Fragments; C071878:S-(4-bromo-2,3-dioxobutyl)glutathione; D005982:Glutathione Transferase; D005978:Glutathione