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

Four subunits of the cytosolic glutathione S-transferase (GST) in Orth osia gothica fed on willow leaves and a semisynthetic bean diet were p urified as separate peaks (subunits 1-4) by a two-step gradient elutio n from a reverse-phase HPLC column after an initial purification by gl utathione-Sepharose affinity chromatography, The reconstituted GST hom odimers all demonstrated activity towards 1-chloro-2,4-dinitro-benzene (CDNB). Subunit 1 with a molecular weight of 26.0 kDa reconstituted i nto a GST homodimer with an isoelectric point of 4.8, and the N-termin al amino acid sequence (27 steps) indicated a relationship to the clas s theta GST of Musca domestica in the first 10 steps (50% homology), b ut also to the GST class pi of Caenohrabditis elegans (50% between ste ps 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) a nd was identical for the three subunits, A high identity of sequence t o the GST in C, elegans (70% between steps 1 and 17), and a low homolo gy (25%) to the O. gothica subunit 1 was observed, Thus, we suggest th e O. gothica subunit 1 belong to a different class (O, gothica GST cla ss 1) of GST than subunits 2-4 (O. gothica GST class 2), When the larv ae 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 incr eased compared to the corresponding ratio in the larvae which hatched and fed on willow leaves until the third instar.