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 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 subunit 2/subunit 1 ratio increased compared to the corresponding ratio in the larvae which hatched and fed on willow leaves until the third instar.
Egaas
E
E
Norwegian Plant Protection Institute, As, Norway.
Sandvik
M
M
Svendsen
N O
NO
Skaare
J U
JU
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004137:Dinitrochlorobenzene / Q000378:metabolism; D005260:Female; D005982:Glutathione Transferase / Q000032:analysis* / Q000145:classification; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology*; D009578:Nitrobenzenes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004137:Dinitrochlorobenzene; D009578:Nitrobenzenes; C028328:1,2-dichloro-4-nitrobenzene; D005982:Glutathione Transferase