Hepatic glutathione S-transferases in mice fed on a diet containing the anticarcinogenic antioxidant butylated hydroxyanisole: Isolation of mouse glutathione S-transferase heterodimers by gradient elution of the glutathione-Sepharose affinity matrix

J D Hayes, L A Kerr, S D Peacock, A D Cronshaw, L I McLellan

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    Induction of glutathione S-transferases (GSTs) is believed to represent an important mechanism whereby butylated hydroxyanisole inhibits chemical carcinogenesis. The soluble hepatic GSTs expressed by mice fed on normal diets are all homodimers comprising Ya3 (Mr 25,800), Yb1 (Mr 26,400) and Yf (Mr 24,800) subunits. In addition to these constitutively expressed GSTs, we have identified enzymes containing Ya1 (Mr 25,600), Ya2 (Mr 25,600), Yb2 (Mr 26,200) and Yb5 (Mr 26,500) subunits from the livers of Balb/c mice fed on diets containing butylated hydroxyanisole (BHA). Gradient affinity elution of GSH-Sepharose has been used to resolve the mouse liver enzymes into several discrete pools of activity from which GSTs were purified by cation-exchange chromatography. The inducible Mu-class Yb2 and Yb5 subunits were separately isolated as the heterodimers GST Yb1Yb2 and GST Yb1Yb5 and their catalytic properties are described; this showed that 1,2-dichloro-4-nitrobenzene and trans-4-phenylbut-3-en-2-one are marker substrates for the mouse Yb1 and Yb2 subunits respectively, but no discriminating model substrate was found that allows the identification of the Yb5 subunit. Individual GST subunits were resolved by reverse-phase h.p.l.c. and their amino acid compositions were determined. Certain subunits (Yb1, Yb2, Yb5 and Yf) were also subjected to automated amino acid sequence analysis, and this demonstrated that the Yb5 subunit has a blocked N-terminus. The mouse Yb1, Yb2 and Yb5 subunits from the major inducible Mu-class heterodimers were cleaved with CNBr and purified peptides from the Yb2 and Yb5 subunits were sequenced. These data show that the Yb2 subunit is distinct from the GSTs that are encoded by the cDNAs that have been cloned from mouse liver cDNA libraries but possesses identity with the protein that is encoded by pmGT2, a cDNA isolated from a mouse fibroblast cell line by Townsend, Goldsmith, Pickett & Cowan [(1989) J. Biol. Chem. 264. 21582-21590]. The sequence data also show that the cDNA encoding the mouse Yb5 subunit has not, to date, been cloned, and the relationship between this subunit and Mu-class GSTs in other species that possess a blocked N-terminus (e.g. rat GST YoYo) is discussed.

    Original languageEnglish
    Pages (from-to)501-12
    Number of pages12
    JournalBiochemical Journal
    Volume277 ( Pt 2)
    Publication statusPublished - 15 Jul 1991



    • Amino Acid Sequence
    • Animals
    • Butylated Hydroxyanisole/pharmacology
    • Chromatography, Affinity/methods
    • Chromatography, High Pressure Liquid
    • Cytosol/enzymology
    • Diet
    • Enzyme Induction
    • Female
    • Glutathione
    • Glutathione Transferase/biosynthesis
    • Humans
    • Isoenzymes/biosynthesis
    • Liver/drug effects
    • Macromolecular Substances
    • Male
    • Mice
    • Mice, Inbred BALB C
    • Molecular Sequence Data
    • Molecular Weight
    • Peptide Fragments/isolation & purification
    • Rats
    • Reference Values
    • Sequence Homology, Nucleic Acid

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