Characterization of a variant rat glutathione S-transferase by cDNA expression in Escherichia coli.
We have isolated a glutathione S-transferase Yb1 subunit cDNA from a lambda gt11 cDNA collection constructed from rat testis poly(A) RNA enriched for glutathione S-transferase mRNA activities. This Yb1 cDNA, designated pGTR201, is identical to our liver Yb1 cDNA clone pGTR200 except for a shorter 5'-untranslated sequence. Active glutathione S-transferase is expressed from this Yb1 cDNA driven by the tac promoter on the plasmid construct pGTR201-KK. The expressed glutathione S-transferase protein begins with the third codon (Met) of the cDNA, and is missing the N-terminal proline of rat liver glutathione S-transferase 3-3. Therefore, our Escherichia coli expressed glutathione S-transferase protein represents a variant form of glutathione S-transferase 3-3 (Yb1Yb1), designated GST 3-3(-1). The expressed Yb1 subunits are assembled into a dimer as purified from sonicated E. coli crude extracts. In the absence of dithiothreitol three active isomers can be resolved by ion-exchange chromatography. The pure protein has an extinction coefficient of 9.21 x 10(4) M-1 cm-1 at 280 nm or E0.1% 280 = 1.78 and a pI at 8.65. It has a substrate specificity pattern similar to that of the authentic glutathione S-transferase 3-3. The GST 3-3(-1) has a KM of 202 microM for reduced GSH and of 36 microM for 1-chloro-2,4-dinitrobenzene. The turnover number for this conjugation reaction is 57 s-1. Results of kinetic studies of this reaction with GST 3-3(-1) are consistent with a sequential substrate binding mechanism. We conclude that the first amino acid proline of glutathione S-transferase 3-3 is not essential for enzyme activities.
Lai
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Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Qian
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D000818:Animals; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000032:analysis* / Q000235:genetics; D007525:Isoelectric Focusing; D010957:Plasmids; D051381:Rats; D013379:Substrate Specificity
D004247:DNA; D005982:Glutathione Transferase