Separation and Characterization of Two Phosphorylase Phosphatase Inhibitors from Rabbit Skeletal Muscle

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Abstract
Two heat‐stable and trypsin‐labile inhibitors of phosphorylase phosphatase, designated Inhibitor‐1 and inhibitor‐2, were partially purified from extracts of rabbit skeletal muscle by heating and column chromtoagraphy using DEAF‐cellulose and Bio‐gel P‐60. Inhibitor‐1 exists in an active phosphorylated form and an inactive dephosphorylated form The interconversion of phosphorylated inhibitor‐1 and dephosphorylated inhibitor‐1 is mediated by protein kinase dependent on adenosine 3: 5 ‐monophosphate (cyclic AMP) and a Mn2+‐stimulated phosphoprotein phosphatase. Inhibitory activity of inhibitor‐2 is not influenced by treatment with either the kinase or the Mn2+ stimudated phosphatase. The molecular weights of inhibitor‐1 and inhibitor‐2 estimated by sodium dodecylsulfate‐polyacrylamide gel electrophoresis are 26000 and 33000 respectively. Both inhibitor‐1 and inhibitor‐2 inhibit phosphorylase phosphatase by a mechanism which appears to be non‐competitive with respect to the substrate phosphorylase a. Inhibitor fractions at early stages of purification also inhibit cyclic‐AMP‐dependent histone phosphorylation, but this kinase inhibitory activity resides with a protein moiety which is separable from inhibitor‐I and inhibitor‐2.

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