Molecular cloning and heterologous expression of a cDNA encoding a mouse glutathione S-transferase Yc subunit possessing high catalytic activity for aflatoxin B1-8,9-epoxide

Resistance to the carcinogenic effects of aflatoxin B1 (AFB1) in the mouse is due to the constitutive expression of an Alpha-class glutathione S-transferase (GST), YcYc, with high detoxification activity towards AFB1-8,9-epoxide. A cDNA clone (pmusGST Yc) for a murine GST Yc polypeptide has been isolated. Sequencing has shown the cDNA insert of pmusGST Yc to be 922 bp in length, with an open reading frame of 663 bp that encodes a polypeptide of M(r) 25358. The primary structure of the murine GST Yc subunit predicted by pmusGST Yc is in complete agreement with the partial amino acid sequence of the aflatoxin-metabolizing mouse liver GST described previously [McLellan, Kerr, Cronshaw & Hayes (1991) Biochem. J. 276, 461-469]. A plasmid, termed pKK-musGST Yc, which permits the expression of the murine Yc subunit in Escherichia coli, has been constructed. The murine GST expressed in E. coli was purified and found to be catalytically active towards several GST substrates, including AFB1-8,9-epoxide. This enzyme was also found to possess electrophoretic and immunochemical properties closely similar to those of the GST Yc subunit from mouse liver. However, the GST synthesized in E. coli and the constitutive mouse liver Alpha-class GST exhibited small differences in their chromatographic behaviour during reverse-phase h.p.l.c. Automated Edman degradation revealed alanine to be the N-terminal amino acid in the GST Yc subunit expressed in E. coli, whereas the enzyme in mouse liver possesses a blocked N-terminus. Although sequencing showed that the purified Yc subunit from E. coli lacked the initiator methionine, the amino acid sequence obtained over the first eleven N-terminal residues agreed with that predicted from the cDNA clone, pmusGST Yc. Comparison of the deduced amino acid sequence of the mouse Yc polypeptide with the primary structures of the rat Alpha-class GST enzymes revealed that it is more closely related to the ethoxyquin-induced rat liver Yc2 subunit than to the constitutively expressed rat liver Yc1 subunit. The significance of the fact that both mouse Yc and rat Yc2 exhibit high catalytic activity towards AFB1-8,9-epoxide, whereas rat Yc1 possesses little activity towards this compound, is discussed in terms of structure/function.

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Regulation of mouse glutathione S-transferases by chemoprotectors. Molecular evidence for the existence of three distinct Alpha-class glutathione S-transferase subunits, Ya1, Ya2, and Ya3, in mouse liver

Biochemical Journal ◽

10.1042/bj2760461 ◽

1991 ◽

Vol 276 (2) ◽

pp. 461-469 ◽

Cited By ~ 31

Author(s):

L I McLellan ◽

L A Kerr ◽

A D Cronshaw ◽

J D Hayes

Keyword(s):

Amino Acid ◽

Sequence Analysis ◽

Amino Acid Sequence ◽

Mouse Liver ◽

Normal Diet ◽

Genomic Clone ◽

Ah Receptor ◽

Molecular Evidence ◽

Glutathione S Transferase ◽

Glutathione S Transferases

Liver cytosol from mice fed on a normal diet contains Alpha-class glutathione S-transferase (GST) subunits of Mr 25,800, Mu-class GST subunits of Mr 26,400 and Pi-class GST subunits of Mr 24,800. Feeding female mice with a diet containing the anticarcinogenic antioxidant butylated hydroxyanisole (BHA) causes induction of the constitutively expressed Mu-class and Pi-class subunits. BHA also induces an Alpha-class GST comprising subunits of Mr 25,600, which is not expressed at detectable levels in normal mouse liver [McLellan & Hayes (1989) Biochem. J. 263, 393-402]. Data are now presented that show that administration of the anticarcinogen beta-naphthoflavone (BNF), like BHA, induces the Alpha-class 25,600-Mr subunits but not the constitutive Alpha-class GST with subunits of Mr 25,800. The effects of BNF on expression of hepatic GST were studied in both DBA/2 and C57BL/6 mice; these studies revealed a preferential induction of the Alpha-class 25,600-Mr subunits and of the Pi-class 24,800-Mr subunits in those mice in possession of a functional Ah receptor. The BHA/BNF-inducible Alpha-class GST can be resolved into two separate, non-interconvertible peaks by reverse-phase h.p.l.c. Automated amino acid sequence analysis of CNBr-derived peptides from each of these h.p.l.c.-purified peaks showed that the peaks contained at least two very similar subunits. These have been named Ya1 and Ya2. The amino acid sequence of the Ya1 subunit was compared with sequences deduced from a genomic clone, lambda mYa1 (Daniel, Sharon, Tichauer & Sarid (1987) DNA 6, 317-324], and a cDNA clone, pGT41 [Pearson, Reinhart, Sisk, Anderson & Adler (1988) J. Biol. Chem. 263, 13324-13332]. Our data suggest that the Ya1 subunit represents the subunit encoded by the genomic clone, lambda mYa1. Sequence analysis of the constitutive Alpha-class Ya3 subunit (Mr 25,800) shows that, although it is a member of the same gene family as the Ya1 and Ya2 subunits, it represents a distinct sub-family of Alpha-class GST, containing subunits that are more similar to rat Yc. Our data indicate that, of these Alpha-class GST subunits, the two with Mr 25,600 (Ya1 and Ya2) are selectively induced by BHA or BNF in mouse liver; neither BHA nor BNF induces significantly the GST subunit with Mr 25,800 (Ya3).

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Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)38864-6 ◽

1985 ◽

Vol 260 (24) ◽

pp. 13268-13271 ◽

Cited By ~ 19

Author(s):

G J Ding ◽

A Y Lu ◽

C B Pickett

Keyword(s):

Amino Acid ◽

Sequence Analysis ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

Rat Liver ◽

Cdna Clone ◽

Nucleotide Sequence Analysis ◽

Liver Glutathione ◽

Complete Amino Acid Sequence ◽

Glutathione S Transferases

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Characterization of two plasmid-encoded cefotaximases found in clinical Escherichia coli isolates: CTX-M-65 and a novel enzyme, CTX-M-87

Journal of Medical Microbiology ◽

10.1099/jmm.0.006007-0 ◽

2009 ◽

Vol 58 (6) ◽

pp. 811-815 ◽

Cited By ~ 19

Author(s):

Jun Yin ◽

Jun Cheng ◽

Zhen Sun ◽

Ying Ye ◽

Yu-Feng Gao ◽

...

Keyword(s):

Escherichia Coli ◽

Catalytic Activity ◽

Amino Acid ◽

Amino Acid Sequence ◽

Hydrolytic Activity ◽

E Coli ◽

High Affinity ◽

Extended Spectrum ◽

M 14

Three clinical strains of Escherichia coli (p168, p517 and p667) were collected in 2006 from three hospitals in Anhui Province (China). PCR and DNA sequencing revealed that E. coli p168 carried a novel extended-spectrum β-lactamase (ESBL), which was designated CTX-M-87. The extended-spectrum β-lactamase which was carried by E. coli p517 and E. coli p667 was previously named CTX-M-65. The deduced amino acid sequence of CTX-M-87, with pI 9.1, differed from that of CTX-M-14 by the substitutions Ala77→Val and Pro167→Leu. Like CTX-M-14, CTX-M-87 had a more potent hydrolytic activity against cefotaxime than against ceftazidime and had high affinity for cefuroxime and cefotaxime. These data show that mutations at position 167 in CTX-M do not always affect catalytic activity and substrate preference.

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