Purification of bile acid-binding proteins from rat hepatic cytosol

Use of a photoaffinity label to detect novel y' binders

Colin J. Henderson, Iain W. Percy-Robb, John D. Hayes

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    A 125I-labelled photolabile derivative of cholic acid has been used to investigate the organic anion-binding Y' fraction from rat liver, prepared by the method of Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1982) Biochimica Biophysica Acta, 709, 342–352. The use of this photoaffinity probe led to the discovery of previously undescribed bile acid-binding proteins. A comprehensive purification scheme for the Y' proteins which allows the isolation of these novel binding species is described. Electrophoretic analysis shows that the Y' binders can be divided into two groups. The proteins in group 1 are dimeric and the 5B, 6E and 7F binding species consist of subunits with approximate molecular masses of 19.6, 15.6 and 14.9 kDa, respectively. The group 2 binding proteins, 5C, 5D and 8C, are monomeric and have molecular masses of approximately 36.2, 36.2 and 33 kDa, respectively. Calculation of the incorporation of 125I by these proteins showed that the group 1 proteins displayed a significantly greater specific incorporation of radioactivity than group 2. The specificity of 125I-labelled 3ß-azidocholylhistamine is further demonstrated by analysis of tryptic digests of photoaffinity labelled Y' binders and glutathione S-transferases AA, A, D and F by reverse-phase high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The majority of the radioactivity was shown to be incorporated into a single component, which was not coincident with the free photoaffinity label.
    Original languageEnglish
    Pages (from-to)270-285
    Number of pages16
    JournalBiochimica et Biophysica Acta. Lipids and Lipid Metabolism
    Volume875
    Issue number2
    DOIs
    Publication statusPublished - 12 Feb 1986

    Keywords

    • Y'-protein
    • Bile acid-binding protein purification
    • Photoaffinity labelling
    • Rat liver

    Cite this

    @article{2f08a61f58324026a51291abb1b43bbd,
    title = "Purification of bile acid-binding proteins from rat hepatic cytosol: Use of a photoaffinity label to detect novel y' binders",
    abstract = "A 125I-labelled photolabile derivative of cholic acid has been used to investigate the organic anion-binding Y' fraction from rat liver, prepared by the method of Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1982) Biochimica Biophysica Acta, 709, 342–352. The use of this photoaffinity probe led to the discovery of previously undescribed bile acid-binding proteins. A comprehensive purification scheme for the Y' proteins which allows the isolation of these novel binding species is described. Electrophoretic analysis shows that the Y' binders can be divided into two groups. The proteins in group 1 are dimeric and the 5B, 6E and 7F binding species consist of subunits with approximate molecular masses of 19.6, 15.6 and 14.9 kDa, respectively. The group 2 binding proteins, 5C, 5D and 8C, are monomeric and have molecular masses of approximately 36.2, 36.2 and 33 kDa, respectively. Calculation of the incorporation of 125I by these proteins showed that the group 1 proteins displayed a significantly greater specific incorporation of radioactivity than group 2. The specificity of 125I-labelled 3{\ss}-azidocholylhistamine is further demonstrated by analysis of tryptic digests of photoaffinity labelled Y' binders and glutathione S-transferases AA, A, D and F by reverse-phase high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The majority of the radioactivity was shown to be incorporated into a single component, which was not coincident with the free photoaffinity label.",
    keywords = "Y'-protein, Bile acid-binding protein purification, Photoaffinity labelling, Rat liver",
    author = "Henderson, {Colin J.} and Percy-Robb, {Iain W.} and Hayes, {John D.}",
    year = "1986",
    month = "2",
    day = "12",
    doi = "10.1016/0005-2760(86)90177-3",
    language = "English",
    volume = "875",
    pages = "270--285",
    journal = "Biochimica et Biophysica Acta. Lipids and Lipid Metabolism",
    issn = "0005-2760",
    publisher = "Elsevier BV",
    number = "2",

    }

    TY - JOUR

    T1 - Purification of bile acid-binding proteins from rat hepatic cytosol

    T2 - Use of a photoaffinity label to detect novel y' binders

    AU - Henderson, Colin J.

    AU - Percy-Robb, Iain W.

    AU - Hayes, John D.

    PY - 1986/2/12

    Y1 - 1986/2/12

    N2 - A 125I-labelled photolabile derivative of cholic acid has been used to investigate the organic anion-binding Y' fraction from rat liver, prepared by the method of Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1982) Biochimica Biophysica Acta, 709, 342–352. The use of this photoaffinity probe led to the discovery of previously undescribed bile acid-binding proteins. A comprehensive purification scheme for the Y' proteins which allows the isolation of these novel binding species is described. Electrophoretic analysis shows that the Y' binders can be divided into two groups. The proteins in group 1 are dimeric and the 5B, 6E and 7F binding species consist of subunits with approximate molecular masses of 19.6, 15.6 and 14.9 kDa, respectively. The group 2 binding proteins, 5C, 5D and 8C, are monomeric and have molecular masses of approximately 36.2, 36.2 and 33 kDa, respectively. Calculation of the incorporation of 125I by these proteins showed that the group 1 proteins displayed a significantly greater specific incorporation of radioactivity than group 2. The specificity of 125I-labelled 3ß-azidocholylhistamine is further demonstrated by analysis of tryptic digests of photoaffinity labelled Y' binders and glutathione S-transferases AA, A, D and F by reverse-phase high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The majority of the radioactivity was shown to be incorporated into a single component, which was not coincident with the free photoaffinity label.

    AB - A 125I-labelled photolabile derivative of cholic acid has been used to investigate the organic anion-binding Y' fraction from rat liver, prepared by the method of Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1982) Biochimica Biophysica Acta, 709, 342–352. The use of this photoaffinity probe led to the discovery of previously undescribed bile acid-binding proteins. A comprehensive purification scheme for the Y' proteins which allows the isolation of these novel binding species is described. Electrophoretic analysis shows that the Y' binders can be divided into two groups. The proteins in group 1 are dimeric and the 5B, 6E and 7F binding species consist of subunits with approximate molecular masses of 19.6, 15.6 and 14.9 kDa, respectively. The group 2 binding proteins, 5C, 5D and 8C, are monomeric and have molecular masses of approximately 36.2, 36.2 and 33 kDa, respectively. Calculation of the incorporation of 125I by these proteins showed that the group 1 proteins displayed a significantly greater specific incorporation of radioactivity than group 2. The specificity of 125I-labelled 3ß-azidocholylhistamine is further demonstrated by analysis of tryptic digests of photoaffinity labelled Y' binders and glutathione S-transferases AA, A, D and F by reverse-phase high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The majority of the radioactivity was shown to be incorporated into a single component, which was not coincident with the free photoaffinity label.

    KW - Y'-protein

    KW - Bile acid-binding protein purification

    KW - Photoaffinity labelling

    KW - Rat liver

    U2 - 10.1016/0005-2760(86)90177-3

    DO - 10.1016/0005-2760(86)90177-3

    M3 - Article

    VL - 875

    SP - 270

    EP - 285

    JO - Biochimica et Biophysica Acta. Lipids and Lipid Metabolism

    JF - Biochimica et Biophysica Acta. Lipids and Lipid Metabolism

    SN - 0005-2760

    IS - 2

    ER -