Expression in Escherichia coli of rat liver cytosolic glutathione S-transferase Yc cDNA

Huskey, Su-Er W.; Wang, Regina W.; Linemeyer, David L.; Pickett, Cecil B.; Lu, Anthony Y. H.

doi:10.1016/0003-9861(90)90470-j

Cited by 5 publications

(3 citation statements)

References 32 publications

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“…Prokaryotic expression of mammalian GSTs has previously been achieved in several cases, including enzymes of the Alpha class [27][28][29], the Mu-class [13,[30][31][32] As a direct way of demonstrating that the cDNAs previously published by Seidegard et al [7] (GTH41 1) and by DeJong et al [13] (GTHb) are encoding functionally similar enzymes and to elucidate whether or not the proteins are identical with GSTs It and if, encoded by the polymorphic GSTI locus, we expressed both these sequences in E. coli.…”

Section: Discussionmentioning

confidence: 99%

Heterologous expression of the allelic variant mu-class glutathione transferases μ and ψ

Widersten

Pearson

Engström

et al. 1991

Biochemical Journal

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cDNA encoding the more acidic form, glutathione transferase (GST) psi, of the polymorphic Mu-class GSTs discovered in liver, was mutated in the 5'-end to create an NcoI site, facilitating cloning into the expression plasmid pKK233-2. The protein expressed from this construct has a point mutation Pro-2----Ala-2, but gives a catalytically functional protein. Back-mutation of the codon for amino acid residue 2 gave rise to a plasmid expressing the wild-type enzyme GST psi, or GST Mu1b-1b. A variant cDNA, differing only in specifying lysine rather than asparagine in position 173 of the coding region, was generated by site-directed mutagenesis. The variant sequence corresponds to another cDNA clone isolated from a human liver cDNA library and expresses the near-neutral GST mu, or GST Mu1a-1a. The two recombinant proteins GST Mu1a-1a and GST Mu1b-1b, by physicochemical as well as kinetic criteria, were found to be indistinguishable from GST mu and GST psi respectively, isolated from human liver. It is therefore concluded that the recombinant proteins correspond to the allelic variants observed in the human population. The two forms have different isoelectric points and correspond to the allelic variants observed in the human population. The two forms have different isoelectric points and their protein subunits can be separated by h.p.l.c. on a reverse-phase column. With standard substrates and inhibitors no differences in kinetic parameters between the two variants were detected. The mutated GST Mu1b-1b (Pro-2----Ala) was not significantly different in catalytic properties from the wild-type enzyme, even though Pro-2 is a well conserved amino acid residue in the known Mu-class GSTs.

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Section: Discussionmentioning

confidence: 99%

Heterologous expression of the allelic variant mu-class glutathione transferases μ and ψ

Widersten

Pearson

Engström

et al. 1991

Biochemical Journal

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“…The availability of vectors for the heterologous expression of the mammalian enzymes in Escherichia coli (24)(25)(26)(27)(28)(29) has encouraged the more the recent application of sitespecific mutagenesis to this problem. Inasmuch as most of this work has yet to appear in the primary literature, only a few highlights will be summarized here.…”

Section: Identification Of Essential Catalytic Residuesmentioning

confidence: 99%

Glutathione S-transferases: reaction mechanism, structure, and function

Armstrong

1991

Chem. Res. Toxicol.

350

253

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“…Western blotting was performed as described (27), using the anti-rat Ya/Yc (1-1/2-2) polyclonal antiserum generously provided by Cecil Pickett (Schering-Plough Research Institute, Kenilworth, NJ). For kinetic analyses, glutathione S-transferase 2-2 was purified essentially as described (28 .01 Cultures of cells that either expressed glutathione S-transferase or did not were grown and induced using the cytotoxicity conditions. Mixtures of equal numbers of cells were treated with solvent or mechlorethamine and plated on agar.…”

Section: Methodsmentioning

confidence: 99%

Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.

Gulick

Fahl

1995

Proc. Natl. Acad. Sci. U.S.A.

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Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze the conjugation, and thus, the detoxication of a structurally diverse group of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards. We proposed that structural alteration of the nonspecific electrophile-binding site would produce mutant enzymes with increased efficiency for detoxication of a single drug and that these mutants could serve as useful somatic transgenes to protect healthy human cells against single alkylating agents used in cancer chemotherapy protocols. Random mutagenesis of three regions (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selection ofEscherichia coli expressing the enzyme library with the nitrogen mustard mechlorethamine (20-500 ,uM), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechlorethamine conjugation (up to 15-fold increase in ka,t and up to 6-fold increase in kct/Km) and that confer up to 31-fold resistance, which is 9-fold greater drug resistance than that conferred by the wild-type enzyme. The results suggest a general strategy for modification of drug-and carcinogen-metabolizing enzymes to achieve desired resistance in both prokaryotic and eukaryotic plant and animal cells.The glutathione S-transferases (EC 2.5.1.18) are a family of enzymes that are responsible for the detoxication of a broad class of electrophiles. In both prokaryotic and eukaryotic cells, these enzymes have been shown to catalyze the conjugation of the tripeptide glutathione to a variety of compounds, resulting in products that are generally less reactive (1-3). High levels of expression of several related glutathione S-transferase isozymes enable these enzymes to protect the cell from numerous structurally diverse electrophiles present in our environment (4-6) and used in cancer chemotherapy (1, 2).Several reports have shown that chronic treatment of cultured cells (7,8) or clinical tumors (9) with alkylating agents results in resistant subpopulations of cells that express alpha class glutathione S-transferases at levels higher than those in the sensitive parental populations. In studies where transfected cDNAs were used to compare cell populations that differed in the single variable of glutathione S-transferase expression, the recombinant rat 1-1 isozyme conferred as much as 3-fold resistance to chlorambucil in mouse fibroblasts (10), and this fold resistance overlaps the fold resistance seen in clinical studies of nitrogen mustard resistance (11,12). Consistent with the observed drug resistance, glutathione S-transferases have been shown to catalyze the conjugation of the nitrogen mustards chlorambucil and melphalan to glutathione (13)(14)(15).Each subunit of a glutathione S-transferase dimer contains an independent active site composed of a G site for binding glutathione and an H site for binding hydrophobic electrophiles (2). Each subunit contain...

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Expression in Escherichia coli of rat liver cytosolic glutathione S-transferase Yc cDNA

Cited by 5 publications

References 32 publications

Heterologous expression of the allelic variant mu-class glutathione transferases μ and ψ

Heterologous expression of the allelic variant mu-class glutathione transferases μ and ψ

Glutathione S-transferases: reaction mechanism, structure, and function

Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.

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