METHODS FOR PURIFICATION OF GLUTATHIONE TRANSFERASES IN THE EARTHWORMGENUS EISENIA, AND THEIR CHARACTERIZATION

Isoenzymes of glutathione transferase (GST) were partially purified fr om the earthworm species Eisenia andrei and E. veneta using affinity c hromatography followed by ion exchange chromatography and reversed-pha se HPLC. In E. veneta, five activity peaks, named EvGST Ia, Ib, II, II I and IV, were separated by anion exchange chromatography. The GSTs in E. andrei were resolved by cation exchange chromatography into six gr oups, named EaGST I-VI. Using reversed-phase HPLC, the affinity-purifi ed GSTs from E. andrei and E. veneta were resolved into 14 subunits, n amed Ea1-Ea14 and Ev1-Ev14, respectively. EaGST I, II, IV and EvGST Ia were further characterized. These forms displayed different substrate specificity towards the substrates 1-chloro-2,4-dinitrobenzene (CDNB) , 1,2-dichloro-4-nitrobenzene, ethacrynic acid (ETHA) and cumene hydro peroxide, as well as different subunit composition determined by SDS-P AGE and reversed-phase HPLC. EaGST IV and EvGST Ia showed exceptionall y high ETHA activity compared with the other forms. EaGST IV consisted of a homodimeric protein involving subunit Ea6 with an apparent molec ular weight of 26.5 kDa, whereas EvGST Ia is composed of two different subunits (Ev9 and Ev10). Amino acid composition and N-terminal analys is of the first 33 residues of Ea6 indicated that the enzyme is most r elated to the pi class. Subunit Ev10 had 67% identity with Ea6, over t he region sequenced (12 residues), but up to 90% identity with GSTs fr om several nematodes. Exposure of both species to trans-stilbene oxide , 3-methylcholanthrene and phenobarbital for three weeks did not eleva te the activity of GST measured with CDNB and ETHA.