Protein Phosphatase Inhibitor-1 and Inhibitor-2 from Rabbit Skeletal Muscle

Philip Cohen, J. Gordon Foulkes, Charles F.B. Holmes, Gillian A. Nimmo, Nicholas K. Tonks

    Research output: Contribution to journalArticle

    81 Citations (Scopus)

    Abstract

    Protein Phosphatase inhibitor-I becomes inhibitory toward protein phosphatase-I only when it has been phosphorylated by cAMPdependent protein kinase, and is likely to play an important role in the control of glycogen metabolism by epinephrine in skeletal muscle. Inhibitor- 2 is a subunit of the cytosolic form of protein phosphatase-l, termed phosphatase-11. Inhibitor-1 and inhibitor-2 are assayed by their ability to inhibit the dephosphorylation of phosphorylase a catalyzed by the free catalytic subtunit of protein phosphatase-1. They are effective at nanomolar levels, which is similar to the concentration of protein phosphatase-1 in the assays. Consequently, the extent of inhibition decreases as the concentration of protein phosphatase-1 increases and vice versa. It is, therefore, important to standardize the protein phosphatase concentration in the assays. Inhibitor-1 is in its dephosphorylated form at each stage of the preparation and must, therefore, be phosphorylated prior to assay. Inhibitor-2 is also diluted in solution A + 0.03% (w/v) Brij-35 and assayed in an identical manner to inhibitor-l, except that prior phosphorylation with cAMP-dependent protein kinase is omitted, and preincubation with protein phosphatase-1 is for 10 rather than 2 min, before addition of 32p-labeled phosphorylase a.

    Original languageEnglish
    Pages (from-to)427-437
    Number of pages11
    JournalMethods in Enzymology
    Volume159
    Issue numberC
    DOIs
    Publication statusPublished - 1988

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    Protein Phosphatase 1
    Phosphoprotein Phosphatases
    Muscle
    Skeletal Muscle
    Phosphorylase a
    Rabbits
    Assays
    Phosphorylation
    Cyclic AMP-Dependent Protein Kinases
    Glycogen
    Phosphoric Monoester Hydrolases
    Metabolism
    Protein Kinases
    Epinephrine
    protein phosphatase inhibitor-1

    Cite this

    Cohen, Philip ; Gordon Foulkes, J. ; Holmes, Charles F.B. ; Nimmo, Gillian A. ; Tonks, Nicholas K. / Protein Phosphatase Inhibitor-1 and Inhibitor-2 from Rabbit Skeletal Muscle. In: Methods in Enzymology. 1988 ; Vol. 159, No. C. pp. 427-437.
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    abstract = "Protein Phosphatase inhibitor-I becomes inhibitory toward protein phosphatase-I only when it has been phosphorylated by cAMPdependent protein kinase, and is likely to play an important role in the control of glycogen metabolism by epinephrine in skeletal muscle. Inhibitor- 2 is a subunit of the cytosolic form of protein phosphatase-l, termed phosphatase-11. Inhibitor-1 and inhibitor-2 are assayed by their ability to inhibit the dephosphorylation of phosphorylase a catalyzed by the free catalytic subtunit of protein phosphatase-1. They are effective at nanomolar levels, which is similar to the concentration of protein phosphatase-1 in the assays. Consequently, the extent of inhibition decreases as the concentration of protein phosphatase-1 increases and vice versa. It is, therefore, important to standardize the protein phosphatase concentration in the assays. Inhibitor-1 is in its dephosphorylated form at each stage of the preparation and must, therefore, be phosphorylated prior to assay. Inhibitor-2 is also diluted in solution A + 0.03{\%} (w/v) Brij-35 and assayed in an identical manner to inhibitor-l, except that prior phosphorylation with cAMP-dependent protein kinase is omitted, and preincubation with protein phosphatase-1 is for 10 rather than 2 min, before addition of 32p-labeled phosphorylase a.",
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    Protein Phosphatase Inhibitor-1 and Inhibitor-2 from Rabbit Skeletal Muscle. / Cohen, Philip; Gordon Foulkes, J.; Holmes, Charles F.B.; Nimmo, Gillian A.; Tonks, Nicholas K.

    In: Methods in Enzymology, Vol. 159, No. C, 1988, p. 427-437.

    Research output: Contribution to journalArticle

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