Covalent labeling of the nonsubstrate ligand-binding site of glutathione S-transferases with bilirubin-Woodward's reagent K

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Abstract

The dimeric enzyme glutathione S-transferase B is composed of two dissimilar subunits, referred to as Y(a) and Y(c). Transferase Y(a)Y(c) and the Y(a)Y(a) homodimer were purified from rat liver cytosol. An enol ester derivative of bilirubin (bilirubin-Woodward's reagent K) was prepared and used to label covalently the nonsubstrate ligand-binding site on these two proteins. There was a linear relationship between the amount of bilirubin-Woodward's reagent K added to the reaction mixture and the amount of labeling achieved up to a ratio of 2:1 (bilirubin-Woodward's reagent K: protein-Y(a)Y(c)). A maximum of 0.87 mol of label bound per mol of transferase Y(a)Y(c). At higher molar ratios, the label appeared to also be binding at a second site on the enzyme. The label blocked the nonsubstrate ligand-binding site of the two transferases but not the catalytic site. The divalent reagent was shown to label equally the Y(a) and Y(c) subunits of transferase Y(a)Y(c), suggesting that the single high affinity bilirubin-binding site present on this protein is formed by an interaction between the subunits rather than residing on a specific subunit. At low ratios of label to protein, bilirubin-Woodward's reagent K appears to label specifically the nonsubstrate ligand-binding site of two forms of glutathione S-transferase, and use of this label should allow for the localization of the nonsubstrate ligand-binding site in the primary amino acid sequence of the Y(a) and Y(c) subunits.

Original languageEnglish (US)
Pages (from-to)5363-5367
Number of pages5
JournalJournal of Biological Chemistry
Volume261
Issue number12
StatePublished - Dec 1 1986

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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