Purification and characterization of multiple glutathione S-transferase isozymes from Chironomidae larvae

Glutathione S-transferase (GST) has been implicated in the process of biotr ansformation of polycyclic aromatic hydrocarbons and of other organic pollu tants by Chironomidae larvae. We have purified and characterized GST from c ytosolic fractions of Chironomidae larvae. GST with an M-r of 23 kDa has be en purified to homogeneity from larvae by centrifugation, size exclusion ch romatography on Sephadex G25, and glutathione affinity and anion exchange c hromatography. The purified enzyme exhibited moderate activity towards 1,2- dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, 4-nitropyridine-N-oxi de, p-nitrobenzyl chloride, ethacrynic acid, and cumene hydroperoxide. The enzyme was homogeneous on gel isoelectric focusing and on SDS gel electroph oresis. Its isoelectric point was estimated to be 5.5. The enzyme had a max imum activity at approximately pH 8 and showed activity between 30 and 40 d egreesC. It became inactive at higher temperature (>50 degreesC) for 5 min. The N-terminal sequence analysis of the amino acids shows a high % of cons erved regions in the enzyme. The enzyme activity was comparable to levels o f metabolism observed by animal GST involved in the detoxification of xenob iotics. (C) 2001 Elsevier Science Inc. All rights reserved.