Sequence of the mRNA for a glutathione transferase Pi with a different substrate specificity in V79 Chinese hamster lung cells.
The mRNA sequence for a glutathione transferase (GST) belonging to the Pi class has been determined. This was a first step towards elucidating, at the molecular level, why V79 Chinese hamster lung cells lack the capacity to conjugate the benzo[a]pyrene (BP) derivative BPDE, but nonetheless contain the GST pi gene, express GST pi mRNA and contain a protein that binds to antibodies directed against the human GST Pi enzyme. The sequencing strategy involved synthesis of a cDNA library, circularization of the GST pi cDNA for PCR amplification and subsequent DNA sequencing. The coding sequence for the GST Pi protein of V79 cells, designated CLOGSTP1, consisted of 627 bp coding for 209 amino acids (aa), corresponding to a 23-kDa protein. The cDNA sequence obtained demonstrated extensive homology to those from other species, especially rat and mouse, where this homology was 92 and 91%, respectively. Upon comparing the aa sequence predicted from CLOGSTP1 to those of rat, mouse, pig, cow and man, the most striking differences were found in aa positions 19, 39, 40, 110, 113 and 151. Consequently, the explanation for the lower capacity for GST Pi-catalyzed conjugation in V79 cells, as compared to other species, remains a matter of speculation, since none of these aa positions coincides with positions involved in the xenobiotic substrate-binding site of GST Pi from pig and human or of GST Mu from rat. The most likely candidates for causing the observed change in substrate specificity might be Lys110 and Glu113, which are the altered residues closest to this binding site and which might, thus, exclude BPDE as a substrate for the Chinese hamster enzyme.
Swedmark
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Department of Genetic and Cellular Toxicology, Wallenberg Laboratory, Stockholm University, Sweden.
Jenssen
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D051381:Rats; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger; D005982:Glutathione Transferase