De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line

Karin Werrbach-Perez, J. Regino Perez-Polo

    Research output: Contribution to journalArticle

    4 Citations (Scopus)

    Abstract

    It is an accepted hypothesis that the nerve growth factor protein (NGF) plays an important role in the development of vertebrate sympathetic and sensory ganglia and has effects on some central neurons. The best known NGF species is that isolated from the mouse submaxillary gland, MSG-NGF. MSG-NGF can be isolated as a subunit containing protein, 7S-NGF, made up of three dissimilar subunits called alpha-, beta-, and gamma-NGF. Beta-NGF is the biologically active subunit and its synthesis in vivo and in vitro has been demonstrated. Less is known about the synthesis of the alpha- and gamma-NGF or the assembly of the subunits into the 7S complex. In order to develop a clonal model system for the study of NGF synthesis, processing and secretion, affinity chromatography techniques were applied to cell extracts of S180 mouse sarcoma, a cell line known to synthesize NGF. After incubating S180 cells in35S-Methionine, cell extracts were exposed to antibody directed against alpha-NGF, gamma-NGF or beta-NGF covalently bound to Sepharose beads in order to elute and characterize the desired NGF subunits. Parallel experiments using immunoabsorbed [35S]Methionine-beta-NGF were carried out in the presence or absence of excess NGF, in order to demonstrate the specificity of this procedure. Affinity chromatography with a substrate analogue to arginine ester bound to Sepharose beads was also used to isolate de novo synthesized gamma-NGF. We were able to show that the S180 line synthesized alpha-, beta-, and gamma-NGF indistiguishable from alpha-, beta-, and gamma-NGF isolated from mouse submaxillary gland in terms of antigenic and physicochemical properties, and biological and enzymatic activities. These results are consistent with the hypothesis that NGF is synthesized, assembled and secreted by a single cell type.

    Original languageEnglish (US)
    Pages (from-to)875-883
    Number of pages9
    JournalNeurochemical Research
    Volume12
    Issue number10
    DOIs
    StatePublished - Oct 1987

    Fingerprint

    Sarcoma 180
    Protein Subunits
    Nerve Growth Factor
    Cells
    Cell Line
    Proteins
    Sodium Glutamate
    Affinity chromatography
    Submandibular Gland
    Cell Extracts
    Affinity Chromatography
    Methionine
    Sepharose
    Sensory Ganglia

    Keywords

    • neuronotrophic factors
    • NGF
    • NGF subunits
    • S180 sarcoma

    ASJC Scopus subject areas

    • Neuroscience(all)
    • Biochemistry

    Cite this

    De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line. / Werrbach-Perez, Karin; Perez-Polo, J. Regino.

    In: Neurochemical Research, Vol. 12, No. 10, 10.1987, p. 875-883.

    Research output: Contribution to journalArticle

    Werrbach-Perez, Karin ; Perez-Polo, J. Regino. / De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line. In: Neurochemical Research. 1987 ; Vol. 12, No. 10. pp. 875-883.
    @article{45939b30bada4a3485233779ea9d2830,
    title = "De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line",
    abstract = "It is an accepted hypothesis that the nerve growth factor protein (NGF) plays an important role in the development of vertebrate sympathetic and sensory ganglia and has effects on some central neurons. The best known NGF species is that isolated from the mouse submaxillary gland, MSG-NGF. MSG-NGF can be isolated as a subunit containing protein, 7S-NGF, made up of three dissimilar subunits called alpha-, beta-, and gamma-NGF. Beta-NGF is the biologically active subunit and its synthesis in vivo and in vitro has been demonstrated. Less is known about the synthesis of the alpha- and gamma-NGF or the assembly of the subunits into the 7S complex. In order to develop a clonal model system for the study of NGF synthesis, processing and secretion, affinity chromatography techniques were applied to cell extracts of S180 mouse sarcoma, a cell line known to synthesize NGF. After incubating S180 cells in35S-Methionine, cell extracts were exposed to antibody directed against alpha-NGF, gamma-NGF or beta-NGF covalently bound to Sepharose beads in order to elute and characterize the desired NGF subunits. Parallel experiments using immunoabsorbed [35S]Methionine-beta-NGF were carried out in the presence or absence of excess NGF, in order to demonstrate the specificity of this procedure. Affinity chromatography with a substrate analogue to arginine ester bound to Sepharose beads was also used to isolate de novo synthesized gamma-NGF. We were able to show that the S180 line synthesized alpha-, beta-, and gamma-NGF indistiguishable from alpha-, beta-, and gamma-NGF isolated from mouse submaxillary gland in terms of antigenic and physicochemical properties, and biological and enzymatic activities. These results are consistent with the hypothesis that NGF is synthesized, assembled and secreted by a single cell type.",
    keywords = "neuronotrophic factors, NGF, NGF subunits, S180 sarcoma",
    author = "Karin Werrbach-Perez and Perez-Polo, {J. Regino}",
    year = "1987",
    month = "10",
    doi = "10.1007/BF00966309",
    language = "English (US)",
    volume = "12",
    pages = "875--883",
    journal = "Neurochemical Research",
    issn = "0364-3190",
    publisher = "Springer New York",
    number = "10",

    }

    TY - JOUR

    T1 - De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line

    AU - Werrbach-Perez, Karin

    AU - Perez-Polo, J. Regino

    PY - 1987/10

    Y1 - 1987/10

    N2 - It is an accepted hypothesis that the nerve growth factor protein (NGF) plays an important role in the development of vertebrate sympathetic and sensory ganglia and has effects on some central neurons. The best known NGF species is that isolated from the mouse submaxillary gland, MSG-NGF. MSG-NGF can be isolated as a subunit containing protein, 7S-NGF, made up of three dissimilar subunits called alpha-, beta-, and gamma-NGF. Beta-NGF is the biologically active subunit and its synthesis in vivo and in vitro has been demonstrated. Less is known about the synthesis of the alpha- and gamma-NGF or the assembly of the subunits into the 7S complex. In order to develop a clonal model system for the study of NGF synthesis, processing and secretion, affinity chromatography techniques were applied to cell extracts of S180 mouse sarcoma, a cell line known to synthesize NGF. After incubating S180 cells in35S-Methionine, cell extracts were exposed to antibody directed against alpha-NGF, gamma-NGF or beta-NGF covalently bound to Sepharose beads in order to elute and characterize the desired NGF subunits. Parallel experiments using immunoabsorbed [35S]Methionine-beta-NGF were carried out in the presence or absence of excess NGF, in order to demonstrate the specificity of this procedure. Affinity chromatography with a substrate analogue to arginine ester bound to Sepharose beads was also used to isolate de novo synthesized gamma-NGF. We were able to show that the S180 line synthesized alpha-, beta-, and gamma-NGF indistiguishable from alpha-, beta-, and gamma-NGF isolated from mouse submaxillary gland in terms of antigenic and physicochemical properties, and biological and enzymatic activities. These results are consistent with the hypothesis that NGF is synthesized, assembled and secreted by a single cell type.

    AB - It is an accepted hypothesis that the nerve growth factor protein (NGF) plays an important role in the development of vertebrate sympathetic and sensory ganglia and has effects on some central neurons. The best known NGF species is that isolated from the mouse submaxillary gland, MSG-NGF. MSG-NGF can be isolated as a subunit containing protein, 7S-NGF, made up of three dissimilar subunits called alpha-, beta-, and gamma-NGF. Beta-NGF is the biologically active subunit and its synthesis in vivo and in vitro has been demonstrated. Less is known about the synthesis of the alpha- and gamma-NGF or the assembly of the subunits into the 7S complex. In order to develop a clonal model system for the study of NGF synthesis, processing and secretion, affinity chromatography techniques were applied to cell extracts of S180 mouse sarcoma, a cell line known to synthesize NGF. After incubating S180 cells in35S-Methionine, cell extracts were exposed to antibody directed against alpha-NGF, gamma-NGF or beta-NGF covalently bound to Sepharose beads in order to elute and characterize the desired NGF subunits. Parallel experiments using immunoabsorbed [35S]Methionine-beta-NGF were carried out in the presence or absence of excess NGF, in order to demonstrate the specificity of this procedure. Affinity chromatography with a substrate analogue to arginine ester bound to Sepharose beads was also used to isolate de novo synthesized gamma-NGF. We were able to show that the S180 line synthesized alpha-, beta-, and gamma-NGF indistiguishable from alpha-, beta-, and gamma-NGF isolated from mouse submaxillary gland in terms of antigenic and physicochemical properties, and biological and enzymatic activities. These results are consistent with the hypothesis that NGF is synthesized, assembled and secreted by a single cell type.

    KW - neuronotrophic factors

    KW - NGF

    KW - NGF subunits

    KW - S180 sarcoma

    UR - http://www.scopus.com/inward/record.url?scp=0023611490&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=0023611490&partnerID=8YFLogxK

    U2 - 10.1007/BF00966309

    DO - 10.1007/BF00966309

    M3 - Article

    C2 - 3683737

    AN - SCOPUS:0023611490

    VL - 12

    SP - 875

    EP - 883

    JO - Neurochemical Research

    JF - Neurochemical Research

    SN - 0364-3190

    IS - 10

    ER -