Purification and characterization of a safener-induced glutathione S-transferase from wheat (Triticum aestivum)

The genome of cultivated wheat is hexaploid, and in consequence a large num ber of glutathione S-transferase (GSTs, EC 2.5.1.18) isozymes is expected i n that organism. Wheat CST subunits were first analyzed by reverse-phase hi gh performance liquid chromatography (RP-HPLC). In root and shoot tissues, subunits 4, 8, and 9 were constitutively expressed whereas subunits 2, 3, a nd 5 were inducible by the herbicide safener naphthalic anhydride (NA). Sig nificant differences were observed, however, between the distributions of t hese six major subunits in roots and shoots. A major GST isozyme was purifi ed from the shoots of plants treated by NA. A combination of ammonium sulph ate precipitation, hydrophobic interaction chromatography (HIC) and affinit y chromatography resulted in purification with an apparent yield of 4.6% an d a 48-fold increase in specific activity toward 1-chloro-2,4-dinitrobenzen e (CDNB). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophor esis (SDS-PAGE) showed a single band at 24.5 kDa. Molecular mass estimated by nondenaturing PAGE was 49.5 kDa. These results suggest that the enzyme e xists as a dimer. A pI of 5.2 was determined by native isoelectric focusing (IEF). Analysis by 2-D electrophoresis showed a single spot, with a pi of 5.8-5.9. However, further analysis by RP-HPLC revealed that the two subunit s were different. They were characterized and identified by electrospray io nization mass spectrometry (ESI-MS) as subunits 2 and 3, molecular masses 2 4924 +/- 3 and 24958 +/- 5 Da, respectively. Therefore, GST(2-3) is apparen tly a heterodimer consisting of subunits 2 and 3. Apparent K-M values were 424 mu M for CDNB and 228 mu M for glutathione (GSH). GST(2-3) metabolized the herbicide fluorodifen, and a K-M of 22 mu M was determined for the herb icide.