Purification and biochemical properties of glutathione S-transferase from Oryza sativa

Abstract

A glutathione S-transferase (GST) from Oryza sativa was purified to electrophoretic homogeneity approximately 742-fold with a 16% activity yield by DEAE-Sephacel and glutathione (GSH)-Sepharose column chromatography. The molecular weight of the enzyme was determined to be approximate to 23 000 by SDS-polyacrylamide gel electrophoresis and 48 000 by gel chromatography, indicating a homodimeric structure. The pI value of the enzyme was 6.4 by chromatofocusing using a Mono P column and the optimum pH was 7.5. The enzyme was retained on GSH affinity column and its K-m value for GSH was 0.36 mM. The activity of the enzyme was significantly inhibited by S-hexyl-GSH and S-(2,4-dinitrophenyl)glutathione. The enzyme displayed activity towards 1-chloro-2,4-dinitrobenzene, a general GST substrate and high activities towards 4-nitrophenethyl bromide and 1,2-epoxy-3-(p-nitrophenoxy) propane, a marker substrate for the theta-class GSTs. It also exhibited glutathione peroxidase activity toward cumene hydroperoxide. (C) 1999 Elsevier Science Inc. All rights reserved.