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. 1993 Jul;102(3):803–810. doi: 10.1104/pp.102.3.803

Purification and characterization of a glutathione S-transferase from benoxacor-treated maize (Zea mays).

G P Irzyk 1, E P Fuerst 1
PMCID: PMC158850  PMID: 8278534

Abstract

A glutathione S-transferase (GST) isozyme from maize (Zea mays Pioneer hybrid 3906) treated with the dichloroacetamide herbicide safener benoxacor (CGA-154281) was purified to homogeneity and partially characterized. The enzyme, assayed with metolachlor as a substrate, was purified approximately 200-fold by ammonium sulfate precipitation, anion-exchange chromatography on Mono Q resins, and affinity chromatography on S-hexylglutathione agarose from total GST activity present in etiolated shoots. The purified protein migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) as a single band with a molecular mass of 27 kD. Using nondenaturing PAGE, we determined that the native protein has a molecular mass of about 57 kD and that the protein exists as a dimer. Two-dimensional electrophoresis revealed only a single protein with an isoelectric point of 5.75 and molecular mass of 27 kD. These results further suggest that the protein exists as a homodimer of two identical 27-kD subunits. The enzyme was most active with substrates possessing a chloroacetamide structure. trans-Cinnamic acid and 1-chloro-2,4-dinitrobenzene were not effective substrates. Apparent Km values for the enzyme were 10.8 microM for the chloroacetamide metolachlor and 292 microM for glutathione. The enzyme was active from pH 6 to 9, with a pH optimum between 7.5 and 8. An apparently blocked amino terminus of the intact protein prevented direct amino acid sequencing. The enzyme was digested with trypsin, and the amino acid sequences of several peptide fragments were obtained. The sequence information for the isolated GST we have designated "GST IV" indicates that the enzyme is a unique maize GST but shares some homology with maize GSTs I and III.

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Selected References

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  1. Boyland E., Chasseaud L. F. The role of glutathione and glutathione S-transferases in mercapturic acid biosynthesis. Adv Enzymol Relat Areas Mol Biol. 1969;32:173–219. doi: 10.1002/9780470122778.ch5. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Clark A. G. The comparative enzymology of the glutathione S-transferases from non-vertebrate organisms. Comp Biochem Physiol B. 1989;92(3):419–446. doi: 10.1016/0305-0491(89)90114-4. [DOI] [PubMed] [Google Scholar]
  4. Dean J. V., Gronwald J. W., Eberlein C. V. Induction of glutathione s-transferase isozymes in sorghum by herbicide antidotes. Plant Physiol. 1990 Feb;92(2):467–473. doi: 10.1104/pp.92.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dudler R., Hertig C., Rebmann G., Bull J., Mauch F. A pathogen-induced wheat gene encodes a protein homologous to glutathione-S-transferases. Mol Plant Microbe Interact. 1991 Jan-Feb;4(1):14–18. doi: 10.1094/mpmi-4-014. [DOI] [PubMed] [Google Scholar]
  6. Fuerst E. P., Irzyk G. P., Miller K. D. Partial Characterization of Glutathione S-Transferase Isozymes Induced by the Herbicide Safener Benoxacor in Maize. Plant Physiol. 1993 Jul;102(3):795–802. doi: 10.1104/pp.102.3.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Grove G., Zarlengo R. P., Timmerman K. P., Li N. Q., Tam M. F., Tu C. P. Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family. Nucleic Acids Res. 1988 Jan 25;16(2):425–438. doi: 10.1093/nar/16.2.425. [DOI] [PMC free article] [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. Hedrick J. L., Smith A. J. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys. 1968 Jul;126(1):155–164. doi: 10.1016/0003-9861(68)90569-9. [DOI] [PubMed] [Google Scholar]
  10. MAQSOOD M., BIOL M. I. Radioisotopes in medicine. Pak J Health. 1957 Jan;6(4):193–203. [PubMed] [Google Scholar]
  11. 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]
  12. Mannervik B., Guthenberg C. Glutathione transferase (human placenta). Methods Enzymol. 1981;77:231–235. doi: 10.1016/s0076-6879(81)77030-7. [DOI] [PubMed] [Google Scholar]
  13. Rabilloud T., Carpentier G., Tarroux P. Improvement and simplification of low-background silver staining of proteins by using sodium dithionite. Electrophoresis. 1988 Jun;9(6):288–291. doi: 10.1002/elps.1150090608. [DOI] [PubMed] [Google Scholar]
  14. Shimabukuro R. H., Swanson H. R., Walsh W. C. Glutathione conjugation: atrazine detoxication mechanism in corn. Plant Physiol. 1970 Jul;46(1):103–107. doi: 10.1104/pp.46.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]

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