[9] Use of antibody- sepharose columns to study hormonal activation of cAMP-dependent protein kinase isozymes

Publisher Summary This chapter presents a procedure using immobilized isoreceptor-specific antibody to assay the amount of cAMP endogenously bound to RI and RII in intact liver tissue or in isolated liver cells. In the first step the pelleted cells are frozen by immersion in liquid N 2 or the liver freeze-clamped in situ and pulverized under liquid nitrogen. The next step is homogenization at subzero temperature. The homogenization medium contains a high concentration of ammonium sulfate to precipitate and stabilize the intracellularly formed complexes of cAMP and R isoproteins. Nucleotides (e.g., cGMP) that do not interfere with the subsequent assay of cAMP are present to block any artifactual binding of cAMP. Ammonium sulfate and glycerol were used to stabilize the endogenously formed complexes. The strategy of the assay is to preserve the intracellularly formed complex of R and cAMP. It thus differs from other assays, which aim at preserving the complex of R and the catalytic subunit of the kinase.