Separation of Two Phosphorylase Kinase Phosphatases from Rabbit Skeletal Muscle

Cyclic‐AMP‐dependent protein kinase catalyses the activation of phosphorylase kinase and the phosphorylation of two serine residues on the α subunit and β subunit of phosphorylase kinase [Cohen, P., Watson, D. C. and Dixon, G. H. (1975)]. The dephosphorylation of phosphorylase kinase has been shown to be catalysed by two distinct enzymes, termed α‐phosphorylase kinase phosphatase and β‐phosphorylase kinase phosphatase. These two enzymes show essentially absolute specificity towards the α and β subunits respectively. The two phosphatases copurified through ethanol fractionation, DEAE‐cellulose chromaiograpity and ammonium sulphate precipitation, but were separated from each other by gel filtration on Sephadex G,200. α‐Phosphorylase kinase phosphatase was purified 500‐fold from the ethanol precipitation step, and β‐phosphorylase kinase phosphatase 320‐fold. The molecular weights estimated by gel filtration were 170‐180000 for α‐phosphorylase kinase phosphatase and 75‐80000 for β‐ phosphorylase kinase phosphatase. Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the β subunit (Cohen, P. (1974)), β‐phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. α‐Phosphorylase kinase phosphatase is an enzyme activity that has not been recognised previously. Since the role of the a‐subunit phosphorylation is to stimulate the rate‐of dephosphorylation of the β subunit (Cohen, P. (1974)), α‐phosphorylase kinase phosphatase can be regarded as the enzyme which inhibits the reversal of the activation of phosphorylase kinase. The implications of these findings for the hormonal control of phosphorylase kinase activity by multisite phosphorylation are discussed.