Purification and Characterization of a Protein Inhibitor of Adenosine 3',5'-Monophosphate-dependent Protein Kinases

A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian cells.

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“…4). The value for the protein kinase inhibitor is very similar to that obtained previously (Walsh et al, 1971).…”

Section: Resultssupporting

confidence: 88%

Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle

Cohen¹,

Nimmo²,

Antoniw³

1977

Biochemical Journal

115

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“…Reconstituted GABAA receptor preparations were prepared from pig cerebral cortex as described previously [3] except that vesicles were formed by dialysis against 20 mM-Tris/HCl (pH 7.5)/150 mM-NaCI/0.02 %0 (w/v) NaN3. ATP, bovine heart protein kinase catalytic subunit (1 unit transfers 1.0 pmol of phosphate from ATP to casein/min at pH 6.5, 30°C) and cyclic AMP-dependent protein kinase inhibitor (Type III) [13] were from Sigma.…”

Section: Experimental Materialsmentioning

confidence: 99%

Phosphorylation of γ-aminobutyrate (GABA)/benzodiazepine receptors by cyclic AMP-dependent protein kinase

Kirkness

Bovenkerk

Ueda

et al. 1989

Biochemical Journal

110

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Preparations of gamma-aminobutyrate (GABA)/benzodiazepine receptor from pig cerebral cortex are composed of three major bands of polypeptides (51, 55 and 57 kDa) which are purified in a ratio of approx. 2:1:1 respectively. Treatment of purified receptor preparations with cyclic AMP-dependent protein kinase resulted in major incorporation of 32P into the 55 kDa band only. The maximum incorporation achieved was 0.6 mol of 32P/mol of 55 kDa polypeptide. The phosphorylated receptor subunit (beta-subunit) displays the same apparent Mr as a band labelled irreversibly with the GABA receptor agonist [3H]muscimol. The two nonphosphorylated subunit polypeptides (51 and 57 kDa) are each labelled irreversibly with [3H]flunitrazepam and are recognized by anti-peptide antibodies specific for alpha-subunits.

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“…The enzymic activities of the epididymal fraction increased markedly (approx. 3-fold) following DEAE-Sephadex chromatography (Table 1), indicating that the rat epididymis may contain a protein kinase inhibitor (Walsh et al, 1971; Szmigielski, Alessandro & Costa, 1977) or other interfering substances that were removed by the Chromatographie procedure.…”

Section: Discussionmentioning

confidence: 99%

Purification and properties of cyclic AMP-dependent protein kinase from rat epididymis

Biswas¹,

Majumder²

1982

Reproduction

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Two protein kinases (I and II: EC 2.7.1.37) that show a high degree of substrate specificity for protamine rather than histones, phosvitin and casein were partly purified from rat epididymal tissue. The enzymes were present in the cytosol because greater than 80% of the enzymic activity was recovered in the soluble fraction. The kinases required Mg2+ for activity although Co2+ and Mn2+ were partial substitutes. Zn2+ (1 mM) inhibited nearly completely the activity of the enzymes. Both the kinases showed high affinity for activation with cyclic AMP compared to other cyclic nucleotides. Amino acid analysis of 32P-labelled protamine product revealed that the kinases transfer the terminal phosphate of ATP to serine residues of the protein. The isoenzymes I and II showed certain differences in relation to their hydroxyapatite-chromatography profiles, pH activation profiles, heat sensitivity and Km for ATP and cyclic AMP.

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Purification and Characterization of a Protein Inhibitor of Adenosine 3',5'-Monophosphate-dependent Protein Kinases

Cited by 866 publications

References 20 publications

Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle

Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle

Phosphorylation of γ-aminobutyrate (GABA)/benzodiazepine receptors by cyclic AMP-dependent protein kinase

Purification and properties of cyclic AMP-dependent protein kinase from rat epididymis

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