Biochemical characterization of Drosophila glutathione S-transferases D1 and D21.
The genomic DNA for the two Drosophila genes, gstD1 and gstD21, were engineered for expression in Escherichia coli by polymerase chain reaction using a pair of specially designed primers. This newly designed expression system produced consistently high yields of the recombinant glutathione S-transferases (GSTs), which were purified to electrophoretic homogeneity by S-hexyl-GSH affinity chromatography. Consistent with their differences in size, GST D1 and GST D21 displayed different mobilities on SDS-polyacrylamide gel electrophoresis. Circular dichroism spectrometry revealed some differences in the protein secondary structural organization between the two GST D isozymes. Polyclonal antibodies against GST D1 and GST D21 revealed that they are immunologically distinct from each other. The GST D1 antiserum cross-reacted weakly with GST D21, but the GST D21 antiserum had no detectable cross-reactivity with GST D1. The amino acid sequences of GST D1 and GST D21 have 70% identity. GST D1 is active toward CDNB with 17% of the catalytic efficiency of the human alpha GST121, whereas CDNB is a poor substrate for GST D21. Both GST D1 and GST D21 have similar levels of GSH peroxidase activity against cumene hydroperoxide. Another major difference in substrate specificities between GST D1 and GST D21 is in the activity of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) dehydrochlorinase, which exists only in the GST D1 isozyme. This is the first definitive demonstration that DDT dehydrochlorinase activity is an intrinsic property of a Drosophila GST. Our results suggest that GST D1 may play a role in DDT metabolism in Drosophila.
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802.
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D000818:Animals; D001483:Base Sequence; D002849:Chromatography, Gas; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003634:DDT / Q000494:pharmacology; D017931:DNA Primers; D004330:Drosophila / Q000187:drug effects / Q000201:enzymology*; D004351:Drug Resistance; D004926:Escherichia coli; D017344:Genes, Insect; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013698:Templates, Genetic
D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D003634:DDT; D005982:Glutathione Transferase; D008190:Lyases; C019990:DDT-dehydrochlorinase