The major mechanism whereby cAMP acts as a second messenger of peptide and protein hormones is via the stimulation of the cAMP-dependent protein kinase. A wide variety of mammalian tissues contain a low molecular weight protein that inhibits the cAMP-dependent protein kinase. Inhibition occurs by direct interaction with the catalytic subunit derived from the holoenzyme by cAMP-mediated dissociation. This inhibitor protein has been of particular use in the characterization of the protein kinase but its potential physiological function and role has yet to be established. Circumstantial evidence in favor of a specific function in cAMP action is its presence in non-stimulated tissues (muscle, brain) at a level sufficient to block approximately 20% of the protein kinase and its high affinity for the catalytic subunit (Kd equals 5 nM). We propose to continue to probe the function of the inhibitor by the following approaches: (1) The multiple size and/or shape conformers and the multiple charge isomers will be examined by chemical, physiochemical and immunological procedures in order to evaluate their relationships. (2) The mechanism of inhibition of the catalytic subunit by the inhibitor will be evaluated by kinetic, chemical and physiochemical probes and by comparison with the inhibition of the catalytic subunit by an inhibitor peptide derived by staphylococcal digestion of the inhibitor, and by synthetic peptide adducts containing both the -Arg-Arg-dipeptidyl recognition sequence and an adenine moiety. (3) We will evaluate conditions (hormonal, metabolic) that may modulate the level of inhibitor in cardiac and skeletal muscle. The hormonally-stimulated phosphorylation of phosphorylase kinase, glycogen synthase, membrane and contractile proteins will be determined in perfused heart and isolated diaphragm as a function of the level of inhibitor within tissues in order to establish whether or not a correlation exists, between the level of inhibitor and cAMP-mediated phosphorylation.
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