Characterization of the Interaction of a Protein Inhibitor with Adenosine 3′,5′-Monophosphate-dependent Protein Kinases

Abstract The titrations of the adenosine 3',5'-monophosphate-dependent protein kinase or the catalytic subunit derived therefrom by a heat-stable protein inhibitor are quantitatively identical. Evidence is presented that the inhibitor does not interact with adenosine 3',5'-monophosphate-dependent protein kinase in the absence of the cyclic nucleotide. There is no displacement of the regulatory subunit from the holoenzyme nor is there formation of a stable inhibitor-holoenzyme complex. The interaction of inhibitor and catalytic subunit is shown to be reversible by sucrose gradient ultracentrifugation and by dilution. The results demonstrate that the inhibitor interacts with adenosine 3',5'-monophosphate-dependent protein kinase only after cyclic nucleotide-promoted dissociation.