Separation of Two Phosphorylase Kinase Phosphatase Activities in Rabbit Skeletal Muscle

Abstract

The 20-40-fold activation of phosphorylase kinase catalysed by cyclic AMP-dependent protein kinase in the presence of cyclic AMP, ATP and Mg2+ ions in vitro (Walsh et al., 1968) represents one of the events leading to the stimulation of skeletal muscle glycogenolysis by adrenalin. However, recent experiments have demonstrated that this reaction is far more complex than had originally been supposed, and the information which led to this conclusion is summarized below. (1) Phosphorylase kinase was shown to be formed from three types of polypeptide chain, termed a, band y, and the smallest active species of the enzyme had the structure (aby)4. Incubation of phosphorylase kinase with the cyclic AMP-dependent protein kinase and ATP-Mg led to phosphorylation at two unique serine residueslabyunit. The fist mol rapidly entered the /I subunit and paralleled the rise in activity, whereas the second mol was incorporated five times more slowly into the a subunit after a short lag period, without any further effect on activity (Cohen, 1973; Cohen et al., 1975). (2) After phosphorylation of phosphorylase kinase in vitro, the preparation was shown to contain trace endogenous phosphorylase kinase phosphatase activity capable of dephosphorylating both the a and b subunits (Cohen & Antoniw, 1973). (3) The phosphorylation of the a subunit was found to enhance the rate of dephosphorylation of the b subunit and accompanying inactivation of the enzyme by a factor of 50. The results showed that the reversible activation of phosphorylase kinase was associated exclusively with the reversible phosphorylation of the subunit, but that there might also be a novel mechanism for actively regulating the reversal of hormonal activation, associated with the phosphorylation of the a subunit (Cohen & Antoniw, 1973). (4) Intravenous injection of adrenalin was shown to activate phosphorylase kinase 5-10-fold in skeletal muscle in vivo, and was accompanied by phosphorylation of the same two sites on the a and subunits that were labelled in vitro (Yeaman & Cohen, 1974,1975). This showed that the two functional changes associated with the phosphorylation of each site were control mechanisms that operated in vivo. Here we present an extension of these findings and demonstrate that there are in fact two distinct phosphorylase kinase phosphatases in skeletal muscle, one specific for the a subunit, and one specific for the