Activation of Cardiac Phosphorylase b Kinase

Abstract Nonactivated, partially purified bovine heart phosphorylase b kinase was activated by incubation with adenosine triphosphate and Mg++ ion prior to assay. Adenosine 3',5'-cyclic phosphate increased the rate of activation, but there was no absolute requirement for the cyclic nucleotide. Activation was particularly prominent when the subsequent assay for kinase activity was carried out at pH 7.0, where the enzyme has low activity. Significant activation was also evident when the assay was performed at pH 8.2, where the nonactivated enzyme is partially active. The response to adenosine 3',5'-cyclic phosphate was linear from 1 x 10-8 to 2 x 10-7 m. Other ribonucleoside 3',5'-cyclic phosphates were without effect except at much higher concentrations. Supramaximal doses of epinephrine (2 µg), when injected into perfused rat hearts, caused a modest but significant activation of phosphorylase b kinase as compared with controls. A 5-fold increase in enzyme activity occurred as determined by assaying 30,000 x g supernatant fractions at pH 6.8. Activation was also apparent, but proportionately less, when extracts were assayed at pH 8.2. Activation was characterized by a 3-fold increase in pH 6.8 to 8.2 activity ratios. The activation occurred rapidly, was maximal at 3 sec after epinephrine injection, and rose well ahead of the contractile response, which reached a maximum in 10 sec.