In:
Biochemical Journal, Portland Press Ltd., Vol. 321, No. 2 ( 1997-01-15), p. 531-536
Abstract:
Although the three-dimensional structure of human glutathione transferase (GST) P1Ő1 crystallized with a GSH analogue has been reported, its structure in the non-complexed form has not been determined. Four monoclonal antibodies to GST P1Ő1 were produced to facilitate structural analysis. Of these, one, clone d-1 of IgG2a isotype, dose-dependently inhibited the activity of GST P1Ő1 but did not affect the activities of either GST A1Ő1 or M1Ő1. On immunoblotting, the antibody reacted strongly with GST P1Ő1 and weakly with rat GST-P and mouse GST-II, indicating cross-reactivity with Pi-class forms but preferential reactivity with GST P1Ő1. When GST P1Ő1 and the antibody were incubated in the presence of 60 ƁM GSH, no inhibition of activity was found, whereas 1-chloro-2,4-dinitrobenzene had no effect at concentrations up to 10 ƁM. The binding of GST P1Ő1 to antibody adsorbed to Protein AŐSepharose was also prevented by both 0.1 mM GSH and N-ethylmaleimide treatment. Trypsin digests of GST P1Ő1 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 P1Ő1 in the absence of GSH.
Type of Medium:
Online Resource
ISSN:
0264-6021
,
1470-8728
Language:
English
Publisher:
Portland Press Ltd.
Publication Date:
1997
detail.hit.zdb_id:
1473095-9
SSG:
12
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