Protein‐Bound Adenosine 3′:5′‐Monophosphate in Liver of Glucagon‐Treated Rats

Abstract

The adenosine 3′:5′‐monophosphate (cyclic AMP) fraction bound to high‐affinity proteins of rat liver cytosol was determined by homogenization of the frozen tissue with a charcoal suspension followed by centrifugation. Protein‐bound cyclic AMP in the supernatant appears to represent mainly or exclusively cyclic AMP bound to the regulatory subunit of protein kinases as indicated by its identity with the phosphodiesterase‐resistant cyclic AMP fraction in homogenates and its close correlation with protein kinase activation in vivo . Parallel determination of total cyclic AMP, protein‐bound cyclic AMP and protein kinase activation in the livers of rats sacrificed between 2 and 120 min after glucagon injection made it possible to correlate these parameters at different levels of total cyclic AMP in vivo . In non‐stimulated tissue, total cyclic AMP was 0.7 pmol/mg tissue, about 50 % being present in the form of protein‐bound cyclic AMP. At a total cyclic AMP level of 1.1–1.2 pmol/mg, half‐maximal values for bound cyclic AMP as well as half‐maximal activation of protein kinases were found. This shows not only a close correlation between these two parameters but also indicates that rather small changes in total cyclic AMP are required in vivo to effect binding of cyclic AMP to the regulatory subunit, R, with the concomitant activation of protein kinase. The reliability of protein‐bound cyclic AMP as an indicator of protein kinase activation was further documented in the livers of stressed animals, in which total cyclic AMP was elevated nearly two‐fold while bound cyclic AMP and protein kinase remained at basal values.