Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Jan 15;321(Pt 2):531–536. doi: 10.1042/bj3210531

Epitope mapping of a monoclonal antibody to human glutathione transferase P1-1 the binding of which is inhibited by glutathione.

T Takahata 1, S Tsuchida 1, M Oomura 1, T Matsumoto 1, J Azumi 1, I Hatayama 1, M Hayakari 1, J Kimura 1, I Kakizaki 1, H Kano 1, K Satoh 1, K Sato 1
PMCID: PMC1218101  PMID: 9020891

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (343.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Batist G., Tulpule A., Sinha B. K., Katki A. G., Myers C. E., Cowan K. H. Overexpression of a novel anionic glutathione transferase in multidrug-resistant human breast cancer cells. J Biol Chem. 1986 Nov 25;261(33):15544–15549. [PubMed] [Google Scholar]
  2. Board P. G., Mannervik B. The contribution of the C-terminal sequence to the catalytic activity of GST2, a human alpha-class glutathione transferase. Biochem J. 1991 Apr 1;275(Pt 1):171–174. doi: 10.1042/bj2750171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dirr H., Reinemer P., Huber R. X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function. Eur J Biochem. 1994 Mar 15;220(3):645–661. doi: 10.1111/j.1432-1033.1994.tb18666.x. [DOI] [PubMed] [Google Scholar]
  4. Engvall E. Enzyme immunoassay ELISA and EMIT. Methods Enzymol. 1980;70(A):419–439. doi: 10.1016/s0076-6879(80)70067-8. [DOI] [PubMed] [Google Scholar]
  5. Galfre G., Howe S. C., Milstein C., Butcher G. W., Howard J. C. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature. 1977 Apr 7;266(5602):550–552. doi: 10.1038/266550a0. [DOI] [PubMed] [Google Scholar]
  6. García-Sáez I., Párraga A., Phillips M. F., Mantle T. J., Coll M. Molecular structure at 1.8 A of mouse liver class pi glutathione S-transferase complexed with S-(p-nitrobenzyl)glutathione and other inhibitors. J Mol Biol. 1994 Apr 1;237(3):298–314. doi: 10.1006/jmbi.1994.1232. [DOI] [PubMed] [Google Scholar]
  7. Guthenberg C., Mannervik B. Purification of glutathione S-transferases from rat lung by affinity chromatography. Evidence for an enzyme form absent in rat liver. Biochem Biophys Res Commun. 1979 Feb 28;86(4):1304–1310. doi: 10.1016/0006-291x(79)90258-4. [DOI] [PubMed] [Google Scholar]
  8. Habig W. H., Pabst M. J., Jakoby W. B. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974 Nov 25;249(22):7130–7139. [PubMed] [Google Scholar]
  9. Hatayama I., Satoh K., Sato K. A cDNA sequence coding a class pi glutathione S-transferase of mouse. Nucleic Acids Res. 1990 Aug 11;18(15):4606–4606. doi: 10.1093/nar/18.15.4606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hatayama I., Satoh K., Sato K. Developmental and hormonal regulation of the major form of hepatic glutathione S-transferase in male mice. Biochem Biophys Res Commun. 1986 Oct 30;140(2):581–588. doi: 10.1016/0006-291x(86)90771-0. [DOI] [PubMed] [Google Scholar]
  11. Hayes J. D., Pulford D. J. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol. 1995;30(6):445–600. doi: 10.3109/10409239509083491. [DOI] [PubMed] [Google Scholar]
  12. Hoesch R. M., Boyer T. D. Localization of a portion of the active site of two rat liver glutathione S-transferases using a photoaffinity label. J Biol Chem. 1989 Oct 25;264(30):17712–17717. [PubMed] [Google Scholar]
  13. Hsu S. M., Raine L., Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981 Apr;29(4):577–580. doi: 10.1177/29.4.6166661. [DOI] [PubMed] [Google Scholar]
  14. Ji X., Zhang P., Armstrong R. N., Gilliland G. L. The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution. Biochemistry. 1992 Oct 27;31(42):10169–10184. doi: 10.1021/bi00157a004. [DOI] [PubMed] [Google Scholar]
  15. Kano T., Sakai M., Muramatsu M. Structure and expression of a human class pi glutathione S-transferase messenger RNA. Cancer Res. 1987 Nov 1;47(21):5626–5630. [PubMed] [Google Scholar]
  16. King D. S., Fields C. G., Fields G. B. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int J Pept Protein Res. 1990 Sep;36(3):255–266. doi: 10.1111/j.1399-3011.1990.tb00976.x. [DOI] [PubMed] [Google Scholar]
  17. Kodate C., Fukushi A., Narita T., Kudo H., Soma Y., Sato K. Human placental form of glutathione S-transferase (GST-pi) as a new immunohistochemical marker for human colonic carcinoma. Jpn J Cancer Res. 1986 Mar;77(3):226–229. [PubMed] [Google Scholar]
  18. Kong K. H., Nishida M., Inoue H., Takahashi K. Tyrosine-7 is an essential residue for the catalytic activity of human class PI glutathione S-transferase: chemical modification and site-directed mutagenesis studies. Biochem Biophys Res Commun. 1992 Feb 14;182(3):1122–1129. doi: 10.1016/0006-291x(92)91848-k. [DOI] [PubMed] [Google Scholar]
  19. LITTLEFIELD J. W. SELECTION OF HYBRIDS FROM MATINGS OF FIBROBLASTS IN VITRO AND THEIR PRESUMED RECOMBINANTS. Science. 1964 Aug 14;145(3633):709–710. doi: 10.1126/science.145.3633.709. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Lo Bello M., Pastore A., Petruzzelli R., Parker M. W., Wilce M. C., Federici G., Ricci G. Conformational states of human placental glutathione transferase as probed by limited proteolysis. Biochem Biophys Res Commun. 1993 Jul 30;194(2):804–810. doi: 10.1006/bbrc.1993.1893. [DOI] [PubMed] [Google Scholar]
  22. Mannervik B., Danielson U. H. Glutathione transferases--structure and catalytic activity. CRC Crit Rev Biochem. 1988;23(3):283–337. doi: 10.3109/10409238809088226. [DOI] [PubMed] [Google Scholar]
  23. Manoharan T. H., Gulick A. M., Puchalski R. B., Servais A. L., Fahl W. E. Structural studies on human glutathione S-transferase pi. Substitution mutations to determine amino acids necessary for binding glutathione. J Biol Chem. 1992 Sep 15;267(26):18940–18945. [PubMed] [Google Scholar]
  24. Manoharan T. H., Gulick A. M., Reinemer P., Dirr H. W., Huber R., Fahl W. E. Mutational substitution of residues implicated by crystal structure in binding the substrate glutathione to human glutathione S-transferase pi. J Mol Biol. 1992 Jul 20;226(2):319–322. doi: 10.1016/0022-2836(92)90949-k. [DOI] [PubMed] [Google Scholar]
  25. Reinemer P., Dirr H. W., Ladenstein R., Huber R., Lo Bello M., Federici G., Parker M. W. Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution. J Mol Biol. 1992 Sep 5;227(1):214–226. doi: 10.1016/0022-2836(92)90692-d. [DOI] [PubMed] [Google Scholar]
  26. Reinemer P., Dirr H. W., Ladenstein R., Schäffer J., Gallay O., Huber R. The three-dimensional structure of class pi glutathione S-transferase in complex with glutathione sulfonate at 2.3 A resolution. EMBO J. 1991 Aug;10(8):1997–2005. doi: 10.1002/j.1460-2075.1991.tb07729.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sato K. Glutathione transferases as markers of preneoplasia and neoplasia. Adv Cancer Res. 1989;52:205–255. doi: 10.1016/s0065-230x(08)60214-6. [DOI] [PubMed] [Google Scholar]
  28. Satoh K., Kitahara A., Soma Y., Inaba Y., Hatayama I., Sato K. Purification, induction, and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3964–3968. doi: 10.1073/pnas.82.12.3964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shen H., Tsuchida S., Tamai K., Sato K. Identification of cysteine residues involved in disulfide formation in the inactivation of glutathione transferase P-form by hydrogen peroxide. Arch Biochem Biophys. 1993 Jan;300(1):137–141. doi: 10.1006/abbi.1993.1019. [DOI] [PubMed] [Google Scholar]
  30. Sinning I., Kleywegt G. J., Cowan S. W., Reinemer P., Dirr H. W., Huber R., Gilliland G. L., Armstrong R. N., Ji X., Board P. G. Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes. J Mol Biol. 1993 Jul 5;232(1):192–212. doi: 10.1006/jmbi.1993.1376. [DOI] [PubMed] [Google Scholar]
  31. Soma Y., Satoh K., Sato K. Purification and subunit-structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker. Biochim Biophys Acta. 1986 Feb 14;869(3):247–258. doi: 10.1016/0167-4838(86)90064-6. [DOI] [PubMed] [Google Scholar]
  32. Suguoka Y., Kano T., Okuda A., Sakai M., Kitagawa T., Muramatsu M. Cloning and the nucleotide sequence of rat glutathione S-transferase P cDNA. Nucleic Acids Res. 1985 Sep 11;13(17):6049–6057. doi: 10.1093/nar/13.17.6049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tamai K., Satoh K., Tsuchida S., Hatayama I., Maki T., Sato K. Specific inactivation of glutathione S-transferases in class Pi by SH-modifiers. Biochem Biophys Res Commun. 1990 Feb 28;167(1):331–338. doi: 10.1016/0006-291x(90)91769-o. [DOI] [PubMed] [Google Scholar]
  34. Tamai K., Shen H. X., Tsuchida S., Hatayama I., Satoh K., Yasui A., Oikawa A., Sato K. Role of cysteine residues in the activity of rat glutathione transferase P (7-7): elucidation by oligonucleotide site-directed mutagenesis. Biochem Biophys Res Commun. 1991 Sep 16;179(2):790–797. doi: 10.1016/0006-291x(91)91886-h. [DOI] [PubMed] [Google Scholar]
  35. Tew K. D., Bomber A. M., Hoffman S. J. Ethacrynic acid and piriprost as enhancers of cytotoxicity in drug resistant and sensitive cell lines. Cancer Res. 1988 Jul 1;48(13):3622–3625. [PubMed] [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tsuchida S., Maki T., Sato K. Purification and characterization of glutathione transferases with an activity toward nitroglycerin from human aorta and heart. Multiplicity of the human class Mu forms. J Biol Chem. 1990 May 5;265(13):7150–7157. [PubMed] [Google Scholar]
  38. Tsuchida S., Sato K. Glutathione transferases and cancer. Crit Rev Biochem Mol Biol. 1992;27(4-5):337–384. doi: 10.3109/10409239209082566. [DOI] [PubMed] [Google Scholar]
  39. Widersten M., Kolm R. H., Björnestedt R., Mannervik B. Contribution of five amino acid residues in the glutathione-binding site to the function of human glutathione transferase P1-1. Biochem J. 1992 Jul 15;285(Pt 2):377–381. doi: 10.1042/bj2850377. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES