Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle

J. F. Antoniw, H. G. Nimmo, S. J. Yeaman, P. Cohen

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    Abstract

    Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G 200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the α subunit or the β subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel filtration step and were termed I, II and III. Protein phosphatase I, apparent mol.wt. 300,000 was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G 200 column. Protein phosphatase II, apparent mol.wt. 170 000, possessed histone phosphatase activity similar to that of protein phosphatase I. It possessed more than 95% of the activity towards the α subunit of phosphorylase kinase that was recovered from Sephadex G 200. It accounted for 10-15% of the glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activity, but less than 5% of the activity against the β subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G 200. Protein phosphatase III was the most active histone phosphatase. It possesed 95% of the phosphorylase phosphatase and β phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activities recovered from Sephadex G 200. It accounted for less than 5% of the α phosphorylase kinase phosphatase activity. Protein phosphatase III was sometimes eluted from Sephadex G 200 as a species of apparent mol. wt. 75 000 (termed IIIA), sometimes as a species of mol.wt. 46 000 (termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases IIIA and IIIB were identical. These findings, taken with the observation that phosphorylase phosphatase, β phosphorylase kinase phosphatase, glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activities co purified up to the Sephadex G 200 step, suggest that a single protein phosphatase (protein phosphatase III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 162, 435-444] which describes a heat stable protein that is a specific inhibitor of protein phosphatase III.

    Original languageEnglish
    Pages (from-to)423-433
    Number of pages11
    JournalBiochemical Journal
    Volume162
    Issue number2
    DOIs
    Publication statusPublished - 1 Jan 1977

    Fingerprint

    Glycogen Synthase
    Phosphoprotein Phosphatases
    Substrate Specificity
    Glycogen
    Phosphoric Monoester Hydrolases
    Metabolism
    Muscle
    Skeletal Muscle
    Rabbits
    Phosphorylase Phosphatase
    Substrates
    Histones
    Phosphorylase Kinase
    Gel Chromatography
    Gels
    Phosphorylase a
    Glycogenolysis
    DEAE-Cellulose Chromatography
    DEAE-Cellulose
    Phosphoproteins

    Cite this

    @article{c1390607d8c241b9b0614300afc59a79,
    title = "Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle",
    abstract = "Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G 200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the α subunit or the β subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel filtration step and were termed I, II and III. Protein phosphatase I, apparent mol.wt. 300,000 was an active histone phosphatase, but it accounted for only 10-15{\%} of the glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activities and 2-3{\%} of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G 200 column. Protein phosphatase II, apparent mol.wt. 170 000, possessed histone phosphatase activity similar to that of protein phosphatase I. It possessed more than 95{\%} of the activity towards the α subunit of phosphorylase kinase that was recovered from Sephadex G 200. It accounted for 10-15{\%} of the glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activity, but less than 5{\%} of the activity against the β subunit of phosphorylase kinase and 1-2{\%} of the phosphorylase phosphatase activity recovered from Sephadex G 200. Protein phosphatase III was the most active histone phosphatase. It possesed 95{\%} of the phosphorylase phosphatase and β phosphorylase kinase phosphatase activities, and 75{\%} of the glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activities recovered from Sephadex G 200. It accounted for less than 5{\%} of the α phosphorylase kinase phosphatase activity. Protein phosphatase III was sometimes eluted from Sephadex G 200 as a species of apparent mol. wt. 75 000 (termed IIIA), sometimes as a species of mol.wt. 46 000 (termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases IIIA and IIIB were identical. These findings, taken with the observation that phosphorylase phosphatase, β phosphorylase kinase phosphatase, glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activities co purified up to the Sephadex G 200 step, suggest that a single protein phosphatase (protein phosphatase III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 162, 435-444] which describes a heat stable protein that is a specific inhibitor of protein phosphatase III.",
    author = "Antoniw, {J. F.} and Nimmo, {H. G.} and Yeaman, {S. J.} and P. Cohen",
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    Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle. / Antoniw, J. F.; Nimmo, H. G.; Yeaman, S. J.; Cohen, P.

    In: Biochemical Journal, Vol. 162, No. 2, 01.01.1977, p. 423-433.

    Research output: Contribution to journalArticle

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    AU - Antoniw, J. F.

    AU - Nimmo, H. G.

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    N2 - Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G 200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the α subunit or the β subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel filtration step and were termed I, II and III. Protein phosphatase I, apparent mol.wt. 300,000 was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G 200 column. Protein phosphatase II, apparent mol.wt. 170 000, possessed histone phosphatase activity similar to that of protein phosphatase I. It possessed more than 95% of the activity towards the α subunit of phosphorylase kinase that was recovered from Sephadex G 200. It accounted for 10-15% of the glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activity, but less than 5% of the activity against the β subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G 200. Protein phosphatase III was the most active histone phosphatase. It possesed 95% of the phosphorylase phosphatase and β phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activities recovered from Sephadex G 200. It accounted for less than 5% of the α phosphorylase kinase phosphatase activity. Protein phosphatase III was sometimes eluted from Sephadex G 200 as a species of apparent mol. wt. 75 000 (termed IIIA), sometimes as a species of mol.wt. 46 000 (termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases IIIA and IIIB were identical. These findings, taken with the observation that phosphorylase phosphatase, β phosphorylase kinase phosphatase, glycogen synthase phosphatase 1 and glycogen synthase phosphatase 2 activities co purified up to the Sephadex G 200 step, suggest that a single protein phosphatase (protein phosphatase III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 162, 435-444] which describes a heat stable protein that is a specific inhibitor of protein phosphatase III.

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