Separation of Two Phosphorylase Kinase Phosphatases from Rabbit Skeletal Muscle

Abstract

The dephosphorylation of phosphorylase kinase has been shown to be catalysed by two distinct enzymes, termed a-phosphorylase kinase phosphatase and P-phosphorylase kinase phosphatase.These two enzymes show essentially absolute specificity towards the a and p subunits respectively.The two phosphatases copurified through ethanol fractionation, DEAE-cellulose chromatography and ammonium sulphate precipitation, but were separated from each other by gel filtration on Sephadex G-200. a-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and p-phosphorylase kinase phosphatase 320-fold. The molecular weights estimated by gel filtration were 170-180000 for a-phosphorylase kinase phosphatase and 75-80000 for Bphosphorylase kinase phosphatase.Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the p subunit (Cohen, P. (1974)), p-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. a-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 /3 subunit (Cohen, P. (1974)), a-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.

The Hormonal Control of Glycogen Metabolism

“…)-32P]ATP [6]. Protein phosphatase-1 was purified 300 -400-fold, and stored at -20 "C, in 50 mM Tris-HCl/l .O mM EDTA pH 7.0 (25 "C) (buffer A), containing 50 mM mercaptoethanol, 6 mM MnC12 and 50'x glycerol [20]. The catalytic subunit of cyclic-AMP-dependent protein kinase was highly purified by chromatography on CM Sephadex [21].…”

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts.

“…has been observed in membrane vesicles isolated from A-431 cells and from normal and RSV-transformed rat cells (6,24), and this phosphatase activity was inhibited by micromolar concentrations of Zn2+ and was insensitive to F-, an inhibitor of most known phosphatases (1). Vanadate has also been shown to inhibit phosphotyrosine dephosphorylation in A-431 membrane preparations (42), and a similar activity found in extracts from rat liver and muscle was inhibited by Zn2+ but was insensitive to EDTA (22).…”

Discovery of A Ca2+‐and calmodulin‐dependent protein phosphatase

“…Several years ago we reported that extracts of rabbit skeletal muscle contained two different enzymes which dephosphorylated the P-subunit and a-subunit relatively specifically [7]. These enzymes were initially termed fl-phosphorylase kinase phosphatase and a-phosphorylase kinase phosphatase [7], but were subsequently renamed protein phosphatase-1 and protein phosphatase-2 [3].…”

“…These enzymes were initially termed fl-phosphorylase kinase phosphatase and a-phosphorylase kinase phosphatase [7], but were subsequently renamed protein phosphatase-1 and protein phosphatase-2 [3].…”