THE SEPARATION AND IDENTIFICATION OF GLUTATHIONE-S-TRANSFERASE SUBUNITS FROM ORTHOSIA-GOTHICA

Four subunits of the cytosolic glutathione S-transferase (GST) in Orthosia gothica fed on willow leaves and a semisynthetic bean diet were purified as separate peaks (subunits 1-4) by a two-step gradient elution from a reverse-phase HPLC column after an initial purification by glutathione-Sepharose affinity chromatography, The reconstituted GST homodimers all demonstrated activity towards 1-chloro-2,4-dinitro-benzene (CDNB). Subunit 1 with a molecular weight of 26.0 kDa reconstituted into a GST homodimer with an isoelectric point of 4.8, and the N-terminal amino acid sequence (27 steps) indicated a relationship to the class theta GST of Musca domestica in the first 10 steps (50% homology), but also to the GST class pi of Caenohrabditis elegans (50% between steps 10 and 20), The three subunits 2-4 all had a molecular weight of 23.5 kDa and the isoelectric points of the reconstituted homodimers were > 9.0, The N-terminal amino acid sequence was determined (24 steps) and was identical for the three subunits, A high identity of sequence to the GST in C, elegans (70% between steps 1 and 17), and a low homology (25%) to the O. gothica subunit 1 was observed, Thus, we suggest the O. gothica subunit 1 belong to a different class (O, gothica GST class 1) of GST than subunits 2-4 (O. gothica GST class 2), When the larvae hatched and fed on a semisynthetic bean diet, subunits 3 and 4 were not present in the HPLC eluate, and the subunit2/subunit 1 ratio increased compared to the corresponding ratio in the larvae which hatched and fed on willow leaves until the third instar.