Association of glycogenolysis with cardiac sarcoplasmic recticulum: II. Effect of glycogen depletion, deoxycholate solubilization and cardiac ischemia: Evidence for a phosphorylase kinase membrane complex

The glycogenolytic enzymes associated with cardiac sarcoplasmic reticulum have been shown to have two major features which make the munique. The conversion of phosphorylase b to phosphorylase a can take place in the presence of 20 mm eGTA which ordinarily inhibits this reaction by chelating the calcium which is required for the enzyme phosphorylase b kinase of muscle. In addition, inhibitors of “debrancher” enzyme such as Tris also inhibit phosphorylase b activity in the sarcoplasmic reticulum-glycogen complex, but do not inhibit purified phosphorylase. The phosphorylase b to a conversion which occurs through the addition of ATP alone is very rapid, being complete within 5–10 min in normal cardiac sarcoplasmic reticulum and converting approximately 30% of the total phosphorylase activity. DOC solubilization irreversibly perturbed the sarcoplasmic reticulum membrane so that EGTA resistance could not be reconstituted although phosphorylase b to a conversion continued to some extent. Thus, EGTA sensitivity depended, at least in part, on some specific property of the native membrane system. Examination of skeletal muscle sarcoplasmic reticulum from fast, slow and mixed skeletal muscle revealed that phosphorylase b to a conversion occurred, but was EGTA sensitive in a manner similar to that of the solubilized cardiac preparation. Glycogen depletion solubilized phosphorylase and uncoupled it from its activating enzymes. This uncoupling could not be reversed by the addition of glycogen at any concentration. A relationship was demonstrated between the phosphorylase activity in sarcoplasmic reticulum fractions and the glycogen concentration in both normal sarcoplasmic reticulum that was amylase treated, and from sarcoplasmic reticulum which was isolated from ischemic tissue. A correlation between density observed on a sucrose density gradient and glycogen concentration was described. The relationship between phosphorylase and debrancher activity depended only on the presence of glycogen and therefore was not as specific a structural feature. The results suggest that the sarcoplasmic reticulum glycogen complex is highly specific and that specific functional couplings between glycogen and sarcoplasmic reticulum membranes exist. Thus, modulation of the complex could occur through control of glycogenolysis or alterations in membrane conformation, and this complex might represent a link between energy metabolism and excitation-contraction coupling. The glycogenolytic enzymes associated with cardiac sarcoplasmic reticulum have been shown to have two major features which make the munique. The conversion of phosphorylase b to phosphorylase a can take place in the presence of 20 mm eGTA which ordinarily inhibits this reaction by chelating the calcium which is required for the enzyme phosphorylase b kinase of muscle. In addition, inhibitors of “debrancher” enzyme such as Tris also inhibit phosphorylase b activity in the sarcoplasmic reticulum-glycogen complex, but do not inhibit purified phosphorylase. The phosphorylase b to a conversion which occurs through the addition of ATP alone is very rapid, being complete within 5–10 min in normal cardiac sarcoplasmic reticulum and converting approximately 30% of the total phosphorylase activity. DOC solubilization irreversibly perturbed the sarcoplasmic reticulum membrane so that EGTA resistance could not be reconstituted although phosphorylase b to a conversion continued to some extent. Thus, EGTA sensitivity depended, at least in part, on some specific property of the native membrane system. Examination of skeletal muscle sarcoplasmic reticulum from fast, slow and mixed skeletal muscle revealed that phosphorylase b to a conversion occurred, but was EGTA sensitive in a manner similar to that of the solubilized cardiac preparation. Glycogen depletion solubilized phosphorylase and uncoupled it from its activating enzymes. This uncoupling could not be reversed by the addition of glycogen at any concentration. A relationship was demonstrated between the phosphorylase activity in sarcoplasmic reticulum fractions and the glycogen concentration in both normal sarcoplasmic reticulum that was amylase treated, and from sarcoplasmic reticulum which was isolated from ischemic tissue. A correlation between density observed on a sucrose density gradient and glycogen concentration was described. The relationship between phosphorylase and debrancher activity depended only on the presence of glycogen and therefore was not as specific a structural feature. The results suggest that the sarcoplasmic reticulum glycogen complex is highly specific and that specific functional couplings between glycogen and sarcoplasmic reticulum membranes exist. Thus, modulation of the complex could occur through control of glycogenolysis or alterations in membrane conformation, and this complex might represent a link between energy metabolism and excitation-contraction coupling.