Characterization of Protein Kinases from Rabbit Reticulocytes

Four fractions of protein kinase activity were resolved and partially purified from the ribosome-free supernatant fraction of rabbit reticulocytes and erythrocytes by DEAE-cellulose and phosphocellulose chromatography. The protein kinase activities from reticulocytes were studied under a variety of assay conditions, and the enzymes were characterized with respect to the capacity to interact with cyclic adenosine 3' : 5'-monophosphate (cAMP), stimulation by cAMP, the effects of isolated regulatory subunit and heatstable inhibitor protein on activity, substrate specificity, and the effects of monovalent and divalent cations. The enzymes were classified into three types according to previously established criteria (Traugh, J. A., Ashby, C. D., and Walsh, D. A. (1974) Methods Enzymol., in press). Three of the resolved enzyme fractions were found to be cAMP-regulated: two of the enzyme activities were identified as cAMP-dependent holoenzyme consisting of regulatory and catalytic subunits (type I); one activity was the free catalytic subunit of the cAMP-dependent protein kinase (type II). The fourth enzyme fraction was a cAMP-independent protein kinase (type III), with properties similar to phosvitin and casein kinases.