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 transferase (GST) P1-1 crystallized with a GSH analogue has been reported, its structure in the non-complexed form has not been determined. Four monoclonal antibodies to GST P1-1 were produced to facilitate structural analysis. Of these, one, clone d-1 of IgG2a isotype, dose-dependently 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 strongly with GST P1-1 and weakly with rat GST-P and mouse GST-II, indicating cross-reactivity with Pi-class forms but preferential reactivity with GST P1-1. When GST P1-1 and the antibody were incubated in the presence of 60 microM GSH, no inhibition of activity was found, whereas 1-chloro-2,4-dinitrobenzene had no effect at concentrations up to 10 microM. The binding of GST P1-1 to antibody adsorbed to Protein A-Sepharose was also prevented by both 0.1 mM GSH and N-ethylmaleimide treatment. Trypsin digests of GST P1-1 were resolved by HPLC and a peptide that reacted with the antibody was detected by absorption experiments. N-Terminal amino acid sequencing revealed the peptide to be in the C-terminal portion of the enzyme, stretching from amino acid residues 198 to 208. A synthetic peptide of this sequence also absorbed the antibody. These results suggest that both GSH bound to the active site and N-ethylmaleimide bound to the cysteine residue repress antibody binding to the C-terminal region. Thus this antibody may be useful for examining the steric configuration of the C-terminal and other regions of GST P1-1 in the absence of GSH.
Takahata
T
T
Second Department of Biochemistry, Hirosaki University School of Medicine, Japan.
Tsuchida
S
S
Oomura
M
M
Matsumoto
T
T
Azumi
J
J
Hatayama
I
I
Hayakari
M
M
Kimura
J
J
Kakizaki
I
I
Kano
H
H
Satoh
K
K
Sato
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry*; D001666:Binding Sites, Antibody / Q000187:drug effects*; D001667:Binding, Competitive / Q000276:immunology; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology / Q000302:isolation & purification; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005982:Glutathione Transferase; D005978:Glutathione; D005033:Ethylmaleimide