Control of Phosphorylase Activity in a Muscle Glycogen Particle: II. ACTIVATION BY CALCIUM

Abstract

Abstract 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 Ca2+ triggers an immediate of phosphorylase (b to a conversion) resulting from kinase activation; no 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 Ca2+ 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 of kinase in this system requires 2 x 10-6 m free Ca2+ (in contrast to approximately 10-7 m calcium for purified kinase solutions), i.e. the same Ca2+ 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 of endogenous phosphorylase b was produced by Mg-ATP and 10-5 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 α-amylase digestion increased the affinity of phosphorylase kinase for Ca2+ 10-fold to the same level observed with the purified enzyme. Readdition of glycogen reversed this effect. Addition of 1 mm glucose-6-P suppressed the flash activation of phosphorylase both by a direct inhibition of the phosphorylase kinase reaction and, indirectly, by increasing the utilization of ATP. Furthermore, activated phosphorylase produced during the reaction was itself partially inhibited by glucose-6-P indicating that phospho-dephospho hybrids (rather than fully phosphorylated phosphorylase a) had been produced. Such hybrids were generated only in the intact (not in the disrupted) protein-glycogen complex.