This chapter elaborates the radioimmunoassay (RIA) of bovine type II cAMP-dependent protein kinase. The only recognized mechanism by which adenosine 3′,5′-mono-phosphate (cAMP) acts in eukaryotic cells is the activation of cAMP-dependent protein kinases (PK). Homogeneous preparations of cAMP-dependent protein kinases have been isolated from several tissues. These enzymes are composed of four subunits, a regulatory (R) dimer and two catalytic (C) subunits. 2-4 Cyclic AMP activates protein kinases, by binding to the regulatory subunit, and concomitantly stimulating the dissociation of the enzyme into R2(cAMP)4 and active catalytic monomers. Regulatory subunits of bovine cerebral cortex Type II enzymes were purified by a combination of the ion-exchange chromatography on diethylethanolamine (DEAE)-cellulose and affinity chromatography on 8-(6-aminohexyl)amino-cAMP sepharose 4B. The percentage 125I-labeled enzyme bound to antiserum is computed, by dividing, the radioactivity precipitated with antiserum minus the radioactivity precipitated with nonimmune serum, by the total radioactivity added to the incubation. The degree of species cross-reactivity in the RIA varies, depending on the degree of structural homology in the enzyme and the characteristics of the antiserum employed.
ASJC Scopus subject areas
- Molecular Biology