Separation of Two Phosphorylase Kinase Phosphatases from Rabbit Skeletal Muscle

Abstract

Cyclic-AMP-dependent protein kinase [EC 2.7.1.37] catalyzes the activation of phosphorylase kinase [EC 2.7.1.38] and the phosphorylation of 2 serine residues on the .alpha. subunit and .beta. subunit of phosphorylase kinase. The dephosphorylation of phosphorylase was shown to be catalyzed by 2 distinct enzymes, termed .alpha.-phosphorylase kinase phosphatase and .beta.-phosphorylase kinase phosphatase [EC 3.1.3.-]. These 2 enzymes show essentially absolute specificity towards the .alpha. and .beta. subunits, respectively. The 2 phosphatases copurified through ethanol fractionation, DEAE-cellulose chromatography and ammonium sulfate precipitation, but were separated from each other by gel filtration on Sephadex G-200. .alpha.-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and .beta.-phosphorylase kinase phosphatase 320-fold. The MW estimated by gel filtration were 170-180,000 for .alpha.-phosphorylase kinase phosphatase and 75-80,000 for .beta.-phosphorylation kinase phosphatase. Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the .beta. subunit, .beta.-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. .alpha.-Phosphorylase kinase phosphatase is an enzyme activity that has not been recognized previously. Since the role of the .alpha.-subunit phosphorylation is to stimulate the rate of dephosphorylation of the .beta. subunit, .alpha.-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.