Binding of nonsubstrate ligands to the glutathione S-transferases

• Published in: The Journal of biological chemistry Vol. 250; no. 22; pp. 8670 - 8673
• Main Authors: Ketley, J N, Habig, W H, Jakoby, W B
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25-11-1975
• Subjects: Anilino Naphthalenesulfonates; Animals; Binding Sites; Glutathione Transferase - metabolism; Kinetics; Ligands; Liver - enzymology; Mathematics; Protein Binding; Protein Conformation; Rats; Spectrometry, Fluorescence; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)40723-0

Fluorescence spectroscopy and inhibition kinetics were used to quantitate the affinity of nonsubstrate ligands for the rat
liver glutathione S-transferases AA, A, B, and C in the presence of glutahione. The dissociation constants KD, for ligands
such as bilirubin, indocyanine green, and hematin were determined by measuring the decrease in the intrinsic fluorescence
of the proteins attendant on the addition of ligand. A second technique, used for compounds which absorb strongly at the excitation
maxima of tryptophan, was to utilize 8-anilinonaphthalen sulfonate in the formation of protein complex fluorescing at a higher
wavelength. The quenching of this complex allowed the determination of the dissociation constants for ligands such as 3,6-dibromosulfophthalein
and cephalothin. These data indicate that all four proteins bind these ligands but do so with different affinities. The bilirubin-induced
decrease in fluorescence was used to estimate the stoichiometry of binding as 1.2 mol of bilirubin bound/mol of transferase
B and 0.7 mol/mol of transferase C. All of the ligands examine are inhibitors of catalytic activity, as tested in a standard
assay with GSH and 1-chloro-2,4-dinitrobenzene as substrates. From these studies we conclude that these proteins have a broad
specificity not only for their substrates, but for the binding of nonsubstrate ligands as well.