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

Reference ID

7218

Title

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

Source

Plant physiology, 1993, vol. 102, pp. 803-810

Authors (2)

Irzyk-G-P

Fuerst-E-P

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.(ABSTRACT
TRUNCATED AT 250 WORDS)