Covalent labeling of the nonsubstrate ligand-binding site of glutathione S-transferases with bilirubin-Woodward's reagent K.
The dimeric enzyme glutathione S-transferase B is composed of two dissimilar subunits, referred to as Ya and Yc. Transferase YaYc and the YaYa 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-YaYc). A maximum of 0.87 mol of label bound per mol of transferase YaYc. 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 Ya and Yc subunits of transferase YaYc, 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 Ya and Yc subunits.
D000818:Animals; D001663:Bilirubin / Q000378:metabolism*; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism*; D007555:Isoxazoles / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D010080:Oxazoles / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013448:Sulfobromophthalein
D007555:Isoxazoles; D046911:Macromolecular Substances; D010080:Oxazoles; D013448:Sulfobromophthalein; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D005982:Glutathione Transferase; D001663:Bilirubin