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Abstract A modified procedure for purification of the adenosine 3',5'-monophosphate-dependent (cyclic AMP-dependent) protein kinase from rabbit skeletal muscle is described. This procedure results in the separation of the enzyme into two peaks of activity on diethylaminoethylcellulose. No interconversion of these two peaks was observed. Both peaks exhibit dependence on cyclic AMP, but under certain conditions the dependence of Peak I on cyclic AMP can be reduced. The concentration of cyclic AMP needed for half-maximal stimulation is 3 x 10-8 m for Peak I and 1.5 x 10-8 m for Peak II. The binding of cyclic AMP to the protein kinase can be reversed by a number of mild chemical treatments and by gel filtration on Sephadex G-25. The apparent Km for ATP in the presence of 10 mm Mg2+ is approximately 1.5 x 10-5 m in the presence or absence of cyclic AMP for either Peak I or Peak II. For Peak II the apparent Km for casein is 0.9 and 0.6 mg per ml in the presence and absence of cyclic AMP, respectively. This apparent Km is increased to 8 mg per ml when 0.1 m NaCl is present in the reaction mixture. The pH optimum is 6.0 for casein and 6.5 for histone phosphorylation for both enzymes. Peak I contains two components which are dependent on cyclic AMP and separable by sucrose density gradient centrifugation; the sedimentation coefficients of these components are 6.8 S and 4.9 S. Peak II activity sediments as a single peak with a sedimentation coefficient of 4.8 S. In the presence of cyclic AMP the protein kinase activity of Peak I sediments as a single component with a sedimentation coefficient of 3.4 S. The protein kinase activity of Peak II also exhibits a much lower sedimentation coefficient in the presence of cyclic AMP than that shown in the absence of this nucleotide.
Reimann et al. (Thu,) studied this question.