The glycogen‐binding subunit of protein phosphatase‐1g from rabbit skeletal muscle

Further characterisation of its structure and glycogen‐binding properties

Michael J. Hubbard, Philip Cohen

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    Abstract

    The glycogen‐bound form of protein phosphatase‐1 (PP‐1g) was previously purified as a heterodimer composed of a 37‐kDa catalytic (C) subunit and a proteolytically sensitive 103‐kDa glycogen‐binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161‐kDa protein, and that the 103‐kDa species (now termed G′) is itself a product of proteolysis. A second phosphorylation site for cAMP‐dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP‐1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP‐1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen‐protein particles during washing with a variety of solvents. The PP‐1G holoenzyme was released from glycogen‐protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP‐1G. Binding experiments with purified PP‐1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high‐affinity glycogen‐binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP‐1G should be almost entirely bound to glycogen.

    Original languageEnglish
    Pages (from-to)457-465
    Number of pages9
    JournalEuropean Journal of Biochemistry
    Volume180
    Issue number2
    DOIs
    Publication statusPublished - Dec 1989

    Fingerprint

    Protein Subunits
    Glycogen
    Muscle
    Phosphorylation
    Skeletal Muscle
    Rabbits
    Proteolysis
    Holoenzymes
    Ionic strength
    Phosphoric Monoester Hydrolases
    Washing
    Osmolar Concentration
    Protein Kinases
    Serine
    Dilution
    Catalytic Domain
    Proteins
    Degradation
    Experiments

    Cite this

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    abstract = "The glycogen‐bound form of protein phosphatase‐1 (PP‐1g) was previously purified as a heterodimer composed of a 37‐kDa catalytic (C) subunit and a proteolytically sensitive 103‐kDa glycogen‐binding (G) subunit [Str{\aa}hlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161‐kDa protein, and that the 103‐kDa species (now termed G′) is itself a product of proteolysis. A second phosphorylation site for cAMP‐dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP‐1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP‐1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen‐protein particles during washing with a variety of solvents. The PP‐1G holoenzyme was released from glycogen‐protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP‐1G. Binding experiments with purified PP‐1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high‐affinity glycogen‐binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP‐1G should be almost entirely bound to glycogen.",
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    T1 - The glycogen‐binding subunit of protein phosphatase‐1g from rabbit skeletal muscle

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    N2 - The glycogen‐bound form of protein phosphatase‐1 (PP‐1g) was previously purified as a heterodimer composed of a 37‐kDa catalytic (C) subunit and a proteolytically sensitive 103‐kDa glycogen‐binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161‐kDa protein, and that the 103‐kDa species (now termed G′) is itself a product of proteolysis. A second phosphorylation site for cAMP‐dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP‐1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP‐1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen‐protein particles during washing with a variety of solvents. The PP‐1G holoenzyme was released from glycogen‐protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP‐1G. Binding experiments with purified PP‐1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high‐affinity glycogen‐binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP‐1G should be almost entirely bound to glycogen.

    AB - The glycogen‐bound form of protein phosphatase‐1 (PP‐1g) was previously purified as a heterodimer composed of a 37‐kDa catalytic (C) subunit and a proteolytically sensitive 103‐kDa glycogen‐binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161‐kDa protein, and that the 103‐kDa species (now termed G′) is itself a product of proteolysis. A second phosphorylation site for cAMP‐dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP‐1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP‐1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen‐protein particles during washing with a variety of solvents. The PP‐1G holoenzyme was released from glycogen‐protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP‐1G. Binding experiments with purified PP‐1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high‐affinity glycogen‐binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP‐1G should be almost entirely bound to glycogen.

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