PLANT PHYSIOLOGY , Vol 102, Issue 3 803-810, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Purification and Characterization of a Glutathione S-Transferase from Benoxacor-Treated Maize (Zea mays)
G. P. Irzyk and E. P. Fuerst
Department of Crop and Soil Sciences, Washington State University, Pullman, Washington 99164-6420
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 [mu]M for the
chloroacetamide metolachlor and 292 [mu]M 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.