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Protein kinase and phosphatases from human polymorphonuclear leucoytes.

P K Tsung, T Sakamoto, G Weissmann

    The Biochemical Journal
    |March 1, 1975
    PubMed
    Summary

    Human polymorphonuclear leucocytes contain a cyclic AMP-stimulated protein kinase. This study identifies and purifies specific histone phosphatases, revealing their distinct properties and subcellular localization within these immune cells.

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    Area of Science:

    • Biochemistry
    • Cell Biology
    • Enzymology

    Background:

    • Human polymorphonuclear leucocytes (PMNs) possess a cytosol protein kinase activated by cyclic adenosine monophosphate (cAMP) and cyclic inosine monophosphate (cIMP).
    • Understanding the regulation of protein phosphorylation and dephosphorylation in PMNs is crucial for comprehending their cellular functions.

    Purpose of the Study:

    • To characterize the protein kinase and identify specific histone phosphatases in human PMNs.
    • To investigate the properties, substrate specificity, and subcellular localization of these enzymes.

    Main Methods:

    • Enzyme purification using gel filtration, substrate-affinity chromatography (histone-Sepharose 4B), and Sephadex G-150 chromatography.
    • Enzyme activity assays, including stimulation by cyclic nucleotides and metal ions (Mn2+), and inhibition studies (ammonium molybdate).

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  • Subcellular fractionation to determine enzyme localization (cytosol vs. lysosomes).

    • Main Results:

    • A cAMP-sensitive protein kinase holoenzyme (MW 66,000) was identified, dissociating into regulatory (MW 45,000) and catalytic (MW 30,000) subunits.
    • Specific histone phosphatases were resolved from non-specific phosphomonoesterases, with purified enzymes showing apparent molecular weights of 45,000 and 18,700.
    • Cytoplasmic histone phosphatase constituted the majority (69%), with a significant portion (20%) associated with lysosomes; both forms required Mn2+ and had a pH optimum of 6.5-7.0.

    Conclusions:

    • Human PMNs contain distinct cAMP-regulated protein kinase and specific histone phosphatase activities.
    • The identified histone phosphatases exhibit unique biochemical properties and subcellular distributions, suggesting specific roles in PMN function.
    • Cyclic nucleotides modulating the protein kinase do not affect the histone phosphatases, indicating independent regulatory pathways.