Purification and characterization of multiple glutathione S-transferase isozymes from Chironomidae larvae.
Glutathione S-transferase (GST) has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by Chironomidae larvae. We have purified and characterized GST from cytosolic fractions of Chironomidae larvae. GST with an M(r) of 23 kDa has been purified to homogeneity from larvae by centrifugation, size exclusion chromatography on Sephadex G25, and glutathione affinity and anion exchange chromatography. The purified enzyme exhibited moderate activity towards 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, 4-nitropyridine-N-oxide, p-nitrobenzyl chloride, ethacrynic acid, and cumene hydroperoxide. The enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 5.5. The enzyme had a maximum activity at approximately pH 8 and showed activity between 30 and 40 degrees C. It became inactive at higher temperature (>50 degrees C) for 5 min. The N-terminal sequence analysis of the amino acids shows a high % of conserved regions in the enzyme. The enzyme activity was comparable to levels of metabolism observed by animal GST involved in the detoxification of xenobiotics.
Yuen
W K
WK
Department of Biochemistry and Environmental Science program, The Chinese University of Hong Kong, Shatin, Hong Kong.
Ho
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D002683:Chironomidae / Q000201:enzymology*; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D007527:Isoenzymes; D010446:Peptide Fragments; D005982:Glutathione Transferase