J. Borgeraas et al., METHODS FOR PURIFICATION OF GLUTATHIONE TRANSFERASES IN THE EARTHWORMGENUS EISENIA, AND THEIR CHARACTERIZATION, Comparative biochemistry and physiology. Part C, Pharmacology toxicology & endocrinology, 114(2), 1996, pp. 129-140
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.