Localization of a portion of the active site of two rat liver glutathione S-transferases using a photoaffinity label

R. M. Hoesch, Thomas D Boyer

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

The glutathione S-transferases are a family of dimeric enzymes that catalyze the reaction between GSH and a variety of electrophiles. Two closely related isozymes, referred to as Y(a)Y(a) and Y(c)Y(c), were purified from rat liver. A radiolabeled azido derivative of glutathione (S-(p-azidophenacyl)[3H]glutathione) was prepared and used to label covalently the active site of the above two glutathione S-transferases. The noncovalently bound affinity label was a competitive inhibitor of glutathione S-transferase Y(a)Y(a) toward both 1-chloro-2,4-dinitrobenzene and GSH. The covalently labeled enzymes no longer bound to a GSH-affinity column, and covalent labeling was reduced in the presence of GSH and S-(dinitrophenyl)glutathione. These results suggest that the affinity label was binding at the active site. The covalently labeled enzymes were digested with trypsin, and the labeled peptides were purified by HPLC and then sequenced. A single-labeled peptide was identified in the tryptic digest of the Y(a)Y(a) isozyme, whereas two labeled peptides were present in the tryptic digest of Y(c)Y(c). The Y(a) peptide sequence was identical with the published deduced sequence of amino acids between residues 212 and 218 and the sequences of the two peptides purified from Y(c) were identical with the deduced sequence of amino acids between 91 and 110 and 206 and 218. Hence, the Y(a) peptide and the smaller peptide purified from Y(c) came from the same region of the Y(a) and Y(c) subunits. This common region and a second region of the Y(c) subunit appear to form a portion of the active site of these two forms of glutathione S-transferase.

Original languageEnglish (US)
Pages (from-to)17712-17717
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number30
StatePublished - 1989
Externally publishedYes

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Photoaffinity Labels
Glutathione Transferase
Liver
Rats
Catalytic Domain
Peptides
Affinity Labels
Isoenzymes
Glutathione
Amino Acid Sequence
Enzymes
Amino Acids
Dinitrochlorobenzene
Trypsin
Labeling
Labels
High Pressure Liquid Chromatography
Derivatives

ASJC Scopus subject areas

  • Biochemistry

Cite this

Localization of a portion of the active site of two rat liver glutathione S-transferases using a photoaffinity label. / Hoesch, R. M.; Boyer, Thomas D.

In: Journal of Biological Chemistry, Vol. 264, No. 30, 1989, p. 17712-17717.

Research output: Contribution to journalArticle

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