Factors affecting the binding of [3H]adenosine 3':5'-cyclic monophosphate to protein kinase from bovine adrenal cortex.
Inorganic salts, several proteins and traces of protein precipitants were tested to find out by what mechanisms they modulate the binding of cyclic [3H]AMP to protein kinase (ATP-protein phosphotransferase; EC 2.7.1.37). The separation of free and bound cyclic AMP by (NH4)2SO4 precipitation was unaffected by the above agents and was more reliable than the Millipore filtration technique. Several binding sites for cyclic AMP were revealed in adrenal-cortex extract. When this extract was used as binding reagent in an assay for cyclic AMP, the standard curve was distorted in the presence of KCl because the salt affected the different binding sites to a varying extent. At high ionic strenth the protein kinase isoenzyme I dissociated and showed an extraordinarily high affinity for cyclic AMP. Trichloroacetate and perchlorate at very low concentrations were able to dissociate the protein kinase and modulate its binding characteristics as well. A progressive decrease in the cyclic AMP-binding capacity occurred on prolonged incubations. The binding protein was protected against inactivation by 2-mercaptoethanol, EDTA and several proteins. It was more resistant to denaturation when complexed to cyclic AMP. The enhancement of cyclic AMP binding by bovine serum albumin was investigated in some detail and appeared to be a pure stabilizing effect. It is proposed that the competitive-binding assays for cyclic AMP based on protein kinase be conducted at high ionic strength and in the presence of stabilizers (protein, EDTA, 2-mercaptoethanol). The interference from agents that may dissociate the protein kinase or influence its stability will thus be decreased.
D000302:Adrenal Cortex / Q000201:enzymology*; D000311:Adrenal Glands / Q000201:enzymology*; D000418:Albumins; D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D000242:Cyclic AMP / Q000302:isolation & purification / Q000378:metabolism*; D066298:In Vitro Techniques; D009994:Osmolar Concentration; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*
D000418:Albumins; D000242:Cyclic AMP; D011494:Protein Kinases