Purification and characterization of two glutathione S-transferase isozymes from indica-type rice involved in herbicide detoxification

Glutathione S-transferases (GSTs; EC 2.5.1.18) are key metabolic enzymes catalyzing the detoxification of several herbicides in many plants. Rice GSTs catalyze the conjugation of the herbicide pretilachlor with glutathione and this reaction is enhanced following treatment with the safener fenelorim. In our ongoing studies to characterize rice GSTs, we have isolated two GST isozymes from etiolated shouts of Teqing (indica type) rice and purified them by means of fast protein liquid chromatography (FPLC) using the following steps: ammonium sulfate precipitation, phenyl superose HR5/5 column, superose 12 HR gel column, GSTrap affinity column, monoQ column, and MonoP column chromatography. The two GST isozymes were purified to homogeneity and partially characterized. Kinetic analysis, molecular mass analysis by native and SDS-PAGE gel chromatography, isoelectric focusing analysis, optimum pH analysis, substrate specificity analysis, and inhibitor analysis revealed the existence of two different GST isozymes of rice. The first isozyme, designated Os GST I-I, was found to be a constitutively expressed homodimer composed of two copies of the 30-kDa subunit (Os GST I). The second GST isozyme, designated 06 GST II-III, is a heterodimer composed of one 25-kDa (Os GST II) and one 28-kDa (Os GST III) subunit. Constitutive expression of the second isozyme is very low, but its activity is induced considerably by the herbicide safener fenclorim. Os GST II-III appears to play an important role in the safening action of fenclorim, which protects rice against injury caused by the pretilachlor. (C) 2002 Elsevier Science (USA).