Hormonal regulation of adenosine 3',5'-monophosphate-dependent protein kinase.

J. D. Corbin, S. L. Keely, T. R. Soderling, C. R. Park

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    Abstract

    There appear to be two classes of protein kinases in rat heart and adipose tissue, types I and II. Type I elutes from DEAE-cellulose at smaller than 0.1 M NaCl and type II at greater than 0.1 M NaCl. The type I enzyme is more readily dissociated by salt or histone than is the type II enzyme. If the type I kinase is first dissociated by cAMP, the subunits reassociate very slowly at 0 degrees C on removal of the cAMP by Sephadex G-25 chromatography, whereas those of type II reassociate very rapidly. Rat heart contains mostly type I and a small amount of type II enzyme, whereas adipose tissue contains almost exclusively the type II enzyme. The adipose tissue enzyme resembles the heart type II kinase in all of the above properties, although the two enzymes are not identical as indicated by slight differences in elution patterns from DEAE-cellulose columns. Incubation of rat epididymal adipose tissue with low concentrations of epinephrine (0.11 muM) increases glycerol production and the fraction of the protein kinase in the active form (activity ratio). The change in cAMP under these conditions is not statistically significant. The presence of insulin inhibits the epinephrine effect on glycerol production and protein kinase but has no measurable effect on cAMP levels. Incubation of adipose tissue with high epinephrine concentrations (11 muM) increases the cAMP level, the protein kinase activity ratio, and glycerol production. Under these conditions insulin decreases the cAMP level and kinase activity ratio but does not reduce glycerol production. The data suggest that very small changes in the tissue cAMP level, undetectable by the assay method, are magnified during the stepwise activation of glycerol output aided possibly by cooperative effects between cAMP and protein kinase. The procedure developed for determining the state of activation of the cAMP-dependent protein kinase in adipose tissue must be modified by reducing the salt concentration of the buffers in order to carry out similar studies in the heart. This reflects the different types of protein kinase in the two tissues. The addition of charcoal to crude extracts of heart prevents protein kinase activation by added cyclic AMP. Charcoal should therefore prevent any activation that could occur if any sequestered cAMP were released during homogenization. Charcoal addition thereby provides a means to distinguish intracellular cAMP activation of the kinase from that which might occur following cell rupture. If epinephrine-perfused hearts are homogenized in the presence of charcoal, epinephrine stimulation of the protein kinase is only slightly decreased. This indicates that the protein kinase is activated intracellularly by cAMP and suggests that all of the cAMP in the cell is available to the protein kinase; i.e., cAMP is not released during homogenization.

    Original languageEnglish (US)
    Pages (from-to)265-279
    Number of pages15
    JournalAdvances in cyclic nucleotide research
    Volume5
    StatePublished - Jan 1 1975

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    ASJC Scopus subject areas

    • Medicine(all)

    Cite this

    Corbin, J. D., Keely, S. L., Soderling, T. R., & Park, C. R. (1975). Hormonal regulation of adenosine 3',5'-monophosphate-dependent protein kinase. Advances in cyclic nucleotide research, 5, 265-279.