Control of Phosphorylase Activity in a Muscle Glycogen Particle

The regulation of enzymatic activity of several enzymes involved in glycogen breakdown has been investigated in a skeletal muscle fraction containing a protein-glycogen complex and elements of the sarcoplasmic reticulum. In this fraction, phosphorylase is entirely in its inactive b form since phosphorylase kinase itself is totally inactive while phosphorylase phosphatase is fully active. Addition of Mg-ATP and Ca’f triggers an immediate activation of phosphorylase (b to a conversion) resulting from kinase activation; no activation occurs with Mg-ATP alone. As soon as all the ATP has been consumed, phosphorylase a is rapidly reconverted to the b form by the phosphatase, and the over-all process can be repeated many times by successive readditions of ATP; this reaction cycle is referred to as “flash activation” of phosphorylase. The CaZf activation of kinase is reversed by chelation of the metal ion and, therefore, not the result of proteolytic attack by the calcium-dependent kinase-activating factor. Half-maximum activation of kinase in this system requires 2 X lop6 M free Ca2+ (in contrast to approximately lo-’ M calcium for purified kinase solutions), i.e. the same CaZ+ concentration needed to trigger muscle contraction. Activation by Ca2+ resulted in a 13-fold increase in affinity of phosphorylase kinase for phosphorylase b. No evidence was obtained that it was mediated or accompanied by a phosphorylation of phosphorylase kinase. Only slight (20%) and delayed activation of endogenous phosphorylase b was produced by Mg-ATP and lops M cyclic adenosine 3’,5’-monophosphate in the absence of added Ca2+, although a 6-fold increase in kinase activity was measured in the usual assay system (at high dilution of phosphorylase kinase and in the presence of purified phosphorylase b). Disruption of the protein-glycogen complex by Lu-amylase digestion increased the ailinity of phosphorylase kinase for