Purification and characterisation of hepatic glutathione transferases from a herbivorous marsupial, the brushtail possum (Trichosurus vulpecula).
A single glutathione transferase isoenzyme was purified from hepatic cytosol of the brushtail possum and shown to represent 3.6 +/- 0.3% of the total cytosolic protein. Characterisation of the enzyme, termed Possum GST 1-1, indicated that it possessed similar catalytic activity and structural homology with isoenzymes belonging to the alpha class of glutathione transferases. This homodimeric GST exhibited a single band with an apparent molecular mass of 25.4 kDa on sodium dodecyl sulphate-polyacrylamide gels and an apparent pI of 9.8. Inhibition studies demonstrated that Possum GST 1-1 displays binding affinity for a range of inhibitors similar to that shown by alpha class GSTs purified from other mammals. Immunoblot analysis demonstrated immuno-cross reactivity between Possum GST 1-1 and antisera raised against human alpha GST, while this GST did not cross-react with antisera raised against human mu and pi GST. N-terminal sequencing of purified Possum GST 1-1 revealed that the N-terminus of the protein is chemically blocked. Sequence analysis of three internal peptide sequences demonstrated homology with mammalian alpha GSTs. Of particular interest is the significant substrate specificity that Possum GST 1-1 displays with both organic and inorganic hydroperoxides. It is proposed that this substrate specificity is an evolutionary adaptation to a diet high in potentially toxic plant allelochemicals.
Bolton
R M
RM
Key Centre for Applied and Nutritional Toxicology, RMIT-University, Melbourne, Vic., Australia.
Ahokas
J T
JT
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003429:Cross Reactions; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005978:Glutathione / Q000032:analysis / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007106:Immune Sera; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009893:Opossums / Q000378:metabolism*; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D012737:Sex Factors; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D007106:Immune Sera; D005982:Glutathione Transferase; D005978:Glutathione