Protein kinase activity stimulated by adenosine 3' :5'-cyclic monophosphate in synaptic-membrane fragments from ox brain. Inhibition of intrinsic activity by free and membrane-bound calcium ions.
1. Cyclic AMP-stimulated protein kinase activity phosphorylating intrinsic substrates in preparations of synaptic-membrane fragments from ox cerebral cortex was examined in relation to (a) the content of membrane-bound Ca(2+) in the preparations and (b) added Ca(2+) in the assay medium. 2. Centrifugal washing of synaptic-membrane fragments with buffered ethane dioxybis(ethylamine)tetra-acetate solutions decreased bound Ca(2+) from 2.8+/-0.4 (s.d.) to 0.9+/-0.3nmol/mg of protein. In washed preparations basal protein kinase activity was increased by about 40% and the cyclic AMP-stimulated activity by about 15%. Addition of Ca(2+) in the concentration range 5-50mum to the assay medium progressively inhibited the kinase activity of the washed preparations; in this range of Ca(2+) concentration the basal activity was inhibited more than the stimulated activity. 3. In unwashed preparations concentrations of Ca(2+) above 100mum inhibited the cyclic AMP-stimulated activity more than the basal activity. 4. The inhibitory effect of several concentrations of Ca(2+) was examined in relation to cyclic AMP concentration; no evidence for competition between Ca(2+) and cyclic AMP for a site on the enzyme was observed.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology*; D000242:Cyclic AMP / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D047428:Protein Kinase Inhibitors; D013570:Synaptic Membranes / Q000187:drug effects / Q000201:enzymology*
D047428:Protein Kinase Inhibitors; D004533:Egtazic Acid; D000242:Cyclic AMP; D002118:Calcium