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Abstract The transformation of phosphorylase b to a catalyzed by phosphorylase kinase as well as the cyclic 3',5'-AMP-dependent activation of skeletal muscle phosphorylase kinase have been shown in vitro to involve phosphorylation of serine residues in the enzymes. The purpose of the present experiments was to determine whether phosphorylation of these proteins occurred in vivo following the administration of epinephrine. 32P-Pi was given to rabbits to label muscle phosphate compounds. Two hours later leg muscle was rapidly frozen 60 sec after the administration of epinephrine. Phosphorylase b purified from the muscle contained no 32P. The epinephrine-induced formation of phosphorylase a was associated with incorporation of 32P, (0.8 mole per 105 g of enzyme) all of which could be removed by alkaline hydrolysis or incubation with phosphorylase phosphatase. The specific activity of this phosphate corresponded to that of the γ-phosphate of the muscle ATP. No incorporation of 32P into the pyridoxal phosphate prosthetic group of phosphorylase was observed in vivo. In contrast, phosphorylase kinase was found to contain bound phosphate irrespective of its degree of activation. It contained 0.4 to 0.8 mole of phosphate per 105 g of protein that could be hydrolyzed by alkali but not by mild acid. This phosphate exchanged in vivo with 32P in the absence of epinephrine, attaining a specific activity greater than that of the γ-phosphate of muscle ATP 1½ to 3 hours after administration of 32P-Pi to the animals. This suggested that phosphorylase kinase was exposed to a different pool of muscle 32P than phosphorylase. Despite an increase in cyclic 3',5'-AMP concentration and kinase activity in the muscle, epinephrine caused no measurable increase in phosphate incorporation into the enzyme. This may be caused by difficulties in detecting small changes in the degree of phosphorylation during activation in vivo of the kinase.
Mayer et al. (Mon,) studied this question.