Purification and Characterization of a Protein Inhibitor of Adenosine 3',5'-Monophosphate-dependent Protein Kinases

The partial purification and characterization of a factor from skeletal muscle which inhibits the activity of adenosine 3',5'-monophosphate-dependent protein kinases from skeletal muscle, heart, liver, adipose tissue, and brain is described. The inhibitor is stable to heating at 96° and to precipitation with 5%, w/v, of trichloracetic acid, but is assumed to be a protein since it is inactivated by proteolytic enzymes. It has a molecular weight of 26,000 by gel exclusion and an S20,w of 1.5 by sucrose density gradient centrifugation. A kinetic analysis of the effect of the inhibitor on the phosphorylation of casein by skeletal muscle protein kinase indicates that it interacts noncompetitively with respect to ATP, the protein substrate, and adenosine 3',5'-monophosphate. The inhibitor promotes a 5-fold increase in the binding constant of adenosine 3',5'-monophosphate to the protein kinase.