Methods for purification of glutathione transferases in the earthworm genus Eisenia, and their characterization.
Isoenzymes of glutathione transferase (GST) were partially purified from the earthworm species Eisenia andrei and E. veneta using affinity chromatography followed by ion exchange chromatography and reversed-phase HPLC. In E. veneta, five activity peaks, named EvGST Ia, Ib, II, III and IV, were separated by anion exchange chromatography. The GSTs in E. andrei were resolved by cation exchange chromatography into six groups, named EaGST I-VI. Using reversed-phase HPLC, the affinity-purified GSTs from E. andrei and E. veneta were resolved into 14 subunits, named 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 hydroperoxide, as well as different subunit composition determined by SDS-PAGE and reversed-phase HPLC. EaGST IV and EvGST Ia showed exceptionally high ETHA activity compared with the other forms. EaGST IV consisted of a homodimeric protein involving subunit Ea6 with an apparent molecular weight of 26.5 kDa, whereas EvGST Ia is composed of two different subunits (Ev9 and Ev10). Amino acid composition and N-terminal analysis of the first 33 residues of Ea6 indicated that the enzyme is most related to the pi class. Subunit Ev10 had 67% identity with Ea6, over the region sequenced (12 residues), but up to 90% identity with GSTs from several nematodes. Exposure of both species to trans-stilbene oxide, 3-methylcholanthrene and phenobarbital for three weeks did not elevate the activity of GST measured with CDNB and ETHA.
Borgeraas
J
J
Department of Biology, University of Oslo, Norway.
Nilsen
K
K
Stenersen
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification*; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000201:enzymology*; D011506:Proteins / Q000032:analysis; D013379:Substrate Specificity
D000596:Amino Acids; D007527:Isoenzymes; D011506:Proteins; D005982:Glutathione Transferase