Protein Inhibitors of Dog-Liver Phosphorylase Phosphatase Dependent on and Independent of Protein Kinase

Dog liver extracts were shown to contain two kinds of heat-stable protein inhibitors of phosphorylase phosphatase. One type (inhibitor 1) depends upon phosphorylation by protein kinase for its inhibitory activity and can be reversibly inactivated by the action of a phosphatase. The other type (inhibitor 2) is spontaneously active and is not affected by protein kinase or phosphatase. The heat-stable phosphatase inhibitors are distinct from the heat-stable protein kinase inhibitor. Inhibitor 1 appears to be an asymmetrical molecule, characterized on gel filtration by a Stokes radius of 3.4 nm, and in sucrose-density-gradient centrifugation by an s20,w value of 1.6 S. Two forms of the spontaneously active inhibitor 2 (termed α and β) were observed which had different specificities towards different phosphatase preparations. Stokes radii of 4.3 nm and 2.7 nm were found for inhibitor 2α and 2β respectively, but sucrose-density-gradient centrifugation revealed the same s20,w value of 1.6 S. Fractions of high-molecular-weight phosphorylase phosphatase preparations from dog liver cytosol which did not contain measurable heat-stable protein inhibitors, could be dissociated and activated by treatment with ethanol or urea. This would suggest that the high-molecular-weight phosphatase, which is insensitive to the protein inhibitors, does not consist of a complex of catalytic unit(s) and these heat-stable inhibitor proteins.