Isolation and Characterization of Two Adenosine 3′,5′- Monophosphate-Dependent Protein Kinases from Bovine Adrenal Cortex*

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Abstract
Bovine adrenocortical cytosol contains two cAMP-dependent protein kinases separable by diethylaminoethyl- cellulose chromatography. The kinases exhibit behavior characteristic of type I and type II enzymes i.e. the enzyme eluting at 80 mM NaCl is activated by NaCl and histone, whereas the enzyme eluting at 140 mM NaCl is not. In addition, a third cAMP-binding protein eluting from DEAE at 120 mM NaCl has been isolated and tentatively identified as type I regulatory subunit. A method is described for the isolation of the catalytic and regulatory subunits of both enzymes from the same starting material using cGMP to dissociate the enzyme subunits and elute the regulatory subunits from N6-aminoethyl-cAMP-Sepharose 4B. The isolated proteins were homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis. Using this technique, molecular weight values of 44,000 for the catalytic subunits, 51,000 for the cAMP-binding protein and type I regulatory subunit, and 56,000 for the type II regulatory subunit were obtained. Molecular weights obtained by sucrose density gradient centrifugation on the cGMP-dissociated holoenzymes were 95,000 and 101,000 for regulatory subunits from type I and II enzymes and 34,000 and 33,000 for their respective catalytic subunits. The apparent Km and Vmax values for catalytic subunits I and II were similar when histone, casein, or ATP was used as substrate. The Vmax for protamine phosphorylation was 3-fold higher with catalytic subunit II. Phosvitin was not a substrate for either subunit. The cAMP-binding protein and regulatory subunit I were able to recombine with, i.e. inhibit, either catalytic subunit. At a molar ratio of 4:1, inhibition was total. The type II regulatory subunit was considerably less effective. Preference for a particular catalytic subunit was not evident in the case of inhibition by either regulatory subunit or the cAMP-binding protein.