Variation in the expression of Mu-class glutathione S-transferase isoenzymes from human skeletal muscle. Evidence for the existence of heterodimers

A J Hussey, L A Kerr, A D Cronshaw, D J Harrison, J D Hayes

    Research output: Contribution to journalArticle

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    Abstract

    The cytosolic glutathione S-transferases (GST) from human skeletal muscle were purified by a combination of affinity chromatography and anion-exchange chromatography followed by either chromatofocusing or hydroxyapatite chromatography. Pi-class and Mu-class GST, but not Alpha-class GST, were isolated from muscle. In addition to a Pi-class GST subunit, which exists as a homodimer, this tissue also contains a total of three distinct neutral-type Mu-class GST subunits, which hybridize to form homodimers or heterodimers. The neutral-type subunits are referred to as N1-N3 and are defined by the decreasing isoelectric points of the homodimers; GST N1N1, N2N2 and N3N3 have estimated pI values of 6.1, 5.3 and less than 5.0 respectively. SDS/PAGE showed that N1, N2 and N3 have Mr values of 26,700, 26,000 and 26,300 respectively. The N1, N2 and N3 subunits are catalytically distinct, with N1 possessing a high activity for trans-4-phenylbut-3-en-2-one and N2 having high activity with 1,2-dichloro-4-nitrobenzene. In skeletal muscle the expression of the N1 subunit, but not of N2 and N3 subunits, was found to differ from specimen to specimen. The N1 subunit was absent from about 50% of samples examined, and the purification results from two different specimens are presented to illustrate this inter-individual variation. Skeletal muscle from one individual (M1), which did not express N1, contained only GST N2N2, N2N3 and pi, whereas the second sample examined (M2) contained GST N1N2, N2N2 and N2N3 as well as GST pi. N-Terminal amino acid sequence analysis supported the electrophoretic evidence that the N2 subunit in GST N1N2, N2N2 and N2N3 represents the same polypeptide. The peptides obtained from CNBr digests of N2 were subjected separately to automated amino acid sequencing, and the results indicate that N2 is distinct but closely related to the protein encoded by the human Mu-class cDNA clone GTH4 [DeJong, Chang, Whang-Peng, Knutsen & Tu (1988) Nucleic Acids Res. 16, 8541-8554]. GST N2N2 is probably identical with GST 4 [Board, Suzuki & Shaw (1988) Biochim. Biophys. Acta 953, 214-217], as over the 24 N-terminal residues of GST 4 there is complete identity between the two enzymes. Our data suggest that the GST 1 and GST 4 loci are part of the same multi-gene family.

    Original languageEnglish
    Pages (from-to)323-32
    Number of pages10
    JournalBiochemical Journal
    Volume273(Pt 2)
    Publication statusPublished - 15 Jan 1991

    Fingerprint

    Glutathione Transferase
    Isoenzymes
    Muscle
    Skeletal Muscle
    Glutathione S-Transferase pi
    Protein Sequence Analysis
    Chromatography
    Peptides
    Affinity chromatography
    Amino Acids
    Isoelectric Point
    Durapatite
    Affinity Chromatography
    Nucleic Acids
    Anions
    Polyacrylamide Gel Electrophoresis
    Complementary DNA
    Clone Cells
    Purification
    Genes

    Keywords

    • Aged
    • Aged, 80 and over
    • Amino Acid Sequence
    • Chromatography, Affinity
    • Cyanogen Bromide
    • Dinitrochlorobenzene/pharmacology
    • Female
    • Gene Expression
    • Genetic Variation
    • Glutathione Transferase/biosynthesis
    • Humans
    • Isoenzymes/biosynthesis
    • Macromolecular Substances
    • Molecular Sequence Data
    • Molecular Weight
    • Multigene Family
    • Muscles/drug effects
    • Protein Conformation
    • Sequence Homology, Nucleic Acid

    Cite this

    @article{dba3e5adf3bf4874b7ed3f4f35c85b8a,
    title = "Variation in the expression of Mu-class glutathione S-transferase isoenzymes from human skeletal muscle. Evidence for the existence of heterodimers",
    abstract = "The cytosolic glutathione S-transferases (GST) from human skeletal muscle were purified by a combination of affinity chromatography and anion-exchange chromatography followed by either chromatofocusing or hydroxyapatite chromatography. Pi-class and Mu-class GST, but not Alpha-class GST, were isolated from muscle. In addition to a Pi-class GST subunit, which exists as a homodimer, this tissue also contains a total of three distinct neutral-type Mu-class GST subunits, which hybridize to form homodimers or heterodimers. The neutral-type subunits are referred to as N1-N3 and are defined by the decreasing isoelectric points of the homodimers; GST N1N1, N2N2 and N3N3 have estimated pI values of 6.1, 5.3 and less than 5.0 respectively. SDS/PAGE showed that N1, N2 and N3 have Mr values of 26,700, 26,000 and 26,300 respectively. The N1, N2 and N3 subunits are catalytically distinct, with N1 possessing a high activity for trans-4-phenylbut-3-en-2-one and N2 having high activity with 1,2-dichloro-4-nitrobenzene. In skeletal muscle the expression of the N1 subunit, but not of N2 and N3 subunits, was found to differ from specimen to specimen. The N1 subunit was absent from about 50{\%} of samples examined, and the purification results from two different specimens are presented to illustrate this inter-individual variation. Skeletal muscle from one individual (M1), which did not express N1, contained only GST N2N2, N2N3 and pi, whereas the second sample examined (M2) contained GST N1N2, N2N2 and N2N3 as well as GST pi. N-Terminal amino acid sequence analysis supported the electrophoretic evidence that the N2 subunit in GST N1N2, N2N2 and N2N3 represents the same polypeptide. The peptides obtained from CNBr digests of N2 were subjected separately to automated amino acid sequencing, and the results indicate that N2 is distinct but closely related to the protein encoded by the human Mu-class cDNA clone GTH4 [DeJong, Chang, Whang-Peng, Knutsen & Tu (1988) Nucleic Acids Res. 16, 8541-8554]. GST N2N2 is probably identical with GST 4 [Board, Suzuki & Shaw (1988) Biochim. Biophys. Acta 953, 214-217], as over the 24 N-terminal residues of GST 4 there is complete identity between the two enzymes. Our data suggest that the GST 1 and GST 4 loci are part of the same multi-gene family.",
    keywords = "Aged, Aged, 80 and over, Amino Acid Sequence, Chromatography, Affinity, Cyanogen Bromide, Dinitrochlorobenzene/pharmacology, Female, Gene Expression, Genetic Variation, Glutathione Transferase/biosynthesis, Humans, Isoenzymes/biosynthesis, Macromolecular Substances, Molecular Sequence Data, Molecular Weight, Multigene Family, Muscles/drug effects, Protein Conformation, Sequence Homology, Nucleic Acid",
    author = "Hussey, {A J} and Kerr, {L A} and Cronshaw, {A D} and Harrison, {D J} and Hayes, {J D}",
    year = "1991",
    month = "1",
    day = "15",
    language = "English",
    volume = "273(Pt 2)",
    pages = "323--32",
    journal = "Biochemical Journal",
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    Variation in the expression of Mu-class glutathione S-transferase isoenzymes from human skeletal muscle. Evidence for the existence of heterodimers. / Hussey, A J; Kerr, L A; Cronshaw, A D; Harrison, D J; Hayes, J D.

    In: Biochemical Journal, Vol. 273(Pt 2), 15.01.1991, p. 323-32.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Variation in the expression of Mu-class glutathione S-transferase isoenzymes from human skeletal muscle. Evidence for the existence of heterodimers

    AU - Hussey, A J

    AU - Kerr, L A

    AU - Cronshaw, A D

    AU - Harrison, D J

    AU - Hayes, J D

    PY - 1991/1/15

    Y1 - 1991/1/15

    N2 - The cytosolic glutathione S-transferases (GST) from human skeletal muscle were purified by a combination of affinity chromatography and anion-exchange chromatography followed by either chromatofocusing or hydroxyapatite chromatography. Pi-class and Mu-class GST, but not Alpha-class GST, were isolated from muscle. In addition to a Pi-class GST subunit, which exists as a homodimer, this tissue also contains a total of three distinct neutral-type Mu-class GST subunits, which hybridize to form homodimers or heterodimers. The neutral-type subunits are referred to as N1-N3 and are defined by the decreasing isoelectric points of the homodimers; GST N1N1, N2N2 and N3N3 have estimated pI values of 6.1, 5.3 and less than 5.0 respectively. SDS/PAGE showed that N1, N2 and N3 have Mr values of 26,700, 26,000 and 26,300 respectively. The N1, N2 and N3 subunits are catalytically distinct, with N1 possessing a high activity for trans-4-phenylbut-3-en-2-one and N2 having high activity with 1,2-dichloro-4-nitrobenzene. In skeletal muscle the expression of the N1 subunit, but not of N2 and N3 subunits, was found to differ from specimen to specimen. The N1 subunit was absent from about 50% of samples examined, and the purification results from two different specimens are presented to illustrate this inter-individual variation. Skeletal muscle from one individual (M1), which did not express N1, contained only GST N2N2, N2N3 and pi, whereas the second sample examined (M2) contained GST N1N2, N2N2 and N2N3 as well as GST pi. N-Terminal amino acid sequence analysis supported the electrophoretic evidence that the N2 subunit in GST N1N2, N2N2 and N2N3 represents the same polypeptide. The peptides obtained from CNBr digests of N2 were subjected separately to automated amino acid sequencing, and the results indicate that N2 is distinct but closely related to the protein encoded by the human Mu-class cDNA clone GTH4 [DeJong, Chang, Whang-Peng, Knutsen & Tu (1988) Nucleic Acids Res. 16, 8541-8554]. GST N2N2 is probably identical with GST 4 [Board, Suzuki & Shaw (1988) Biochim. Biophys. Acta 953, 214-217], as over the 24 N-terminal residues of GST 4 there is complete identity between the two enzymes. Our data suggest that the GST 1 and GST 4 loci are part of the same multi-gene family.

    AB - The cytosolic glutathione S-transferases (GST) from human skeletal muscle were purified by a combination of affinity chromatography and anion-exchange chromatography followed by either chromatofocusing or hydroxyapatite chromatography. Pi-class and Mu-class GST, but not Alpha-class GST, were isolated from muscle. In addition to a Pi-class GST subunit, which exists as a homodimer, this tissue also contains a total of three distinct neutral-type Mu-class GST subunits, which hybridize to form homodimers or heterodimers. The neutral-type subunits are referred to as N1-N3 and are defined by the decreasing isoelectric points of the homodimers; GST N1N1, N2N2 and N3N3 have estimated pI values of 6.1, 5.3 and less than 5.0 respectively. SDS/PAGE showed that N1, N2 and N3 have Mr values of 26,700, 26,000 and 26,300 respectively. The N1, N2 and N3 subunits are catalytically distinct, with N1 possessing a high activity for trans-4-phenylbut-3-en-2-one and N2 having high activity with 1,2-dichloro-4-nitrobenzene. In skeletal muscle the expression of the N1 subunit, but not of N2 and N3 subunits, was found to differ from specimen to specimen. The N1 subunit was absent from about 50% of samples examined, and the purification results from two different specimens are presented to illustrate this inter-individual variation. Skeletal muscle from one individual (M1), which did not express N1, contained only GST N2N2, N2N3 and pi, whereas the second sample examined (M2) contained GST N1N2, N2N2 and N2N3 as well as GST pi. N-Terminal amino acid sequence analysis supported the electrophoretic evidence that the N2 subunit in GST N1N2, N2N2 and N2N3 represents the same polypeptide. The peptides obtained from CNBr digests of N2 were subjected separately to automated amino acid sequencing, and the results indicate that N2 is distinct but closely related to the protein encoded by the human Mu-class cDNA clone GTH4 [DeJong, Chang, Whang-Peng, Knutsen & Tu (1988) Nucleic Acids Res. 16, 8541-8554]. GST N2N2 is probably identical with GST 4 [Board, Suzuki & Shaw (1988) Biochim. Biophys. Acta 953, 214-217], as over the 24 N-terminal residues of GST 4 there is complete identity between the two enzymes. Our data suggest that the GST 1 and GST 4 loci are part of the same multi-gene family.

    KW - Aged

    KW - Aged, 80 and over

    KW - Amino Acid Sequence

    KW - Chromatography, Affinity

    KW - Cyanogen Bromide

    KW - Dinitrochlorobenzene/pharmacology

    KW - Female

    KW - Gene Expression

    KW - Genetic Variation

    KW - Glutathione Transferase/biosynthesis

    KW - Humans

    KW - Isoenzymes/biosynthesis

    KW - Macromolecular Substances

    KW - Molecular Sequence Data

    KW - Molecular Weight

    KW - Multigene Family

    KW - Muscles/drug effects

    KW - Protein Conformation

    KW - Sequence Homology, Nucleic Acid

    UR - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1149849/

    M3 - Article

    C2 - 1991033

    VL - 273(Pt 2)

    SP - 323

    EP - 332

    JO - Biochemical Journal

    JF - Biochemical Journal

    SN - 0264-6021

    ER -