Cyclic nucleotide-dependent protein kinases. V. Preparation and properties of adenosine 3′,5′-monophosphate-dependent protein kinase from various bovine tissues

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Adenosine 3′,5′-monophosphate (cyclic AMP)_dependent protein kinases, which catalyze the phosphorylation of proteins by ATP, have been found in each of fifteen bovine tissues examined. The enzymes were partially purified from these tissues, and their properties studied. Cyclic AMP increased by 5–15-fold the ability of each of these enzymes to phosphorylate histones. The concentration of cyclic AMP required to give half-maximal activation ranged from 30 to 160 nM. The 3′,5′-monophosphate derivatives of inosine, guanosine, uridine, and cytidine also activated the enzymes, but only at concentrations considerably higher than those required for cyclic AMP. 2′-Deoxythymidine 3′,5′-monophosphate was incapable of stimulating enzyme activity. Of the proteins tested as substrates, histone was the most effective for all of the enzymes studied. Protamine and casein were also phosphorylated by the enzymes. All of the enzymes had an absolute requirement for a divalent metal. Cyclic AMP stimulated enzyme activity in the presence of Mg 2+, Mn 2+, or Co 2+, whereas it inhibited enzyme activity in the presence of Ca 2+. The concentration of ATP required to give half-maximal velocity was determined for several of the enzyme preparations in the absence and presence of cyclic AMP; in each case, the apparent K m for ATP was significantly lower in the presence than in the absence of the activator. It was found that adenine, adenosine, AMP, ADP, GDP, riboflavin, FMN, and FAD inhibited enzyme activity to varying degrees. The fifteen enzyme preparations were found to be generally similar, but some important differences were observed.