EPITOPE MAPPING OF A MONOCLONAL-ANTIBODY TO HUMAN GLUTATHIONE TRANSFERASE P1-1 THE BINDING OF WHICH IS INHIBITED BY GLUTATHIONE

Although the three-dimensional structure of human glutathione transfer ase (GST) P1-1 crystallized with a GSH analogue has been reported, its structure in the non-complexed form has not been determined. Four mon oclonal antibodies to GST P1-1 were produced to facilitate structural analysis. Of these, one, clone d-1 of IgG(2a) isotype, dose-dependentl y inhibited the activity of GST P1-1 but did not affect the activities of either GST A1-1 or M1-1. On immunoblotting, the antibody reacted s trongly with GST P1-1 and weakly with rat GST-P and mouse GST-II, indi cating cross-reactivity with Pi-class forms but preferential reactivit y with GST P1-1. When GST P1-1 and the antibody were incubated in the presence of 60 mu M GSH, no inhibition of activity was found, whereas 1-chloro-2,4-dinitrobenzene had no effect at concentrations up to 10 m u M. The binding of GST P1-1 to antibody adsorbed to Protein A-Sepharo se was also prevented by both 0.1 mM GSH and N-ethylmaleimide treatmen t. Trypsin digests of GST P1-1 were resolved by HPLC and a peptide tha t reacted with the antibody was detected by absorption experiments. N- Terminal amino acid sequencing revealed the peptide to be in the C-ter minal portion of the enzyme, stretching from amino acid residues 198 t o 208. A synthetic peptide of this sequence also absorbed the antibody . These results suggest that both GSH bound to the active site and N-e thylmaleimide bound to the cysteine residue repress antibody binding t o the C-terminal region. Thus this antibody may be useful for examinin g the steric configuration of the C-terminal and other regions of GST P1-1 in the absence of GSH.