Action Patterns of Phosphorylase and Glycogen Synthetase on Glycogen (original) (raw)
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Biochemical journal. Cellular aspects, 1983
The activity of glycogen synthase phosphatase in rat liver stems from the cooperation of two proteins, a cytosolic S-component and a glycogen-bound G-component. It is shown that both components possess synthase phosphatase activity. The G-component was partially purified from the enzyme-glycogen complex. Dissociative treatments, which increase the activity of phosphorylase phosphatase manyfold, substantially decrease the synthase phosphatase activity of the purified G-component. 2. The specific inhibition of glycogen synthase phosphatase by phosphorylase a, originally observed in crude liver extracts, was investigated with purified liver synthase b and purified phosphorylase a. Synthase phosphatase is strongly inhibited, whether present in a dilute liver extract, in an isolated enzyme-glycogen complex, or as G-component purified therefrom. In contrast, the cytosolic S-component is insensitive to phosphorylase a. 3. The activation of glycogen synthase in crude extracts of skeletal muscle is not affected by phosphorylase a from muscle or liver. Consequently we have studied the dephosphorylation of purified muscle glycogen synthase, previously phosphorylated with any of three protein kinases. Phosphorylase a strongly inhibits the dephosphorylation by the hepatic G-component, but not by the hepatic S-component or by a muscle extract. 4. These observations show that the inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.
A titrimetric assay for glycogen phosphorylase
Analytical Biochemistry, 1973
A titrimetric method for the assay of glycogen phosphorylase is presented in which a direct and continuous course of reaction is obtained over a wide range of enzyme concentrations (7.2-378.3 pg/ml). The method resulted in rates which were in agreement with those obtained using the inorganic phosphate method, and the expected value of the equilibrium concentration ratio of inorganic phosphate to glucose-l-phosphate was obtained. The method can be extended to higher concentrations, and it can be used to measure the rate in either direction. The K, and V,., values of each substrate, glucose-l-phosphate and inorganic phosphate, were determined.
FEBS Letters, 2003
Changes in the glucosylation state of the glycogen primer, glycogenin, or its association with glycogen synthase are potential sites for regulation of glycogen synthesis. In this study we found no evidence for hormonal control of the glucosylation state of glycogenin in hepatocytes. However, using a modified glycogen synthase assay that separates the product into acid‐soluble (glycogen) and acid‐insoluble (proteoglycogen) fractions we found that insulin and glucagon increase and decrease, respectively, the association of glycogen synthase with an acid‐insoluble substrate. The latter fraction had a higher affinity for UDP‐glucose and accounted for between 5 and 21% of total activity depending on hormonal conditions. Phosphorylase overexpression mimicked the effect of glucagon. It is concluded that phosphorylase activation or overexpression causes dissociation of glycogen synthase from proteoglycogen causing inhibition of initiation of glycogen synthesis.
European Journal of Biochemistry, 1988
The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3 -5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26"C, but were much more pronounced at the nonpermissive temperature of 35°C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyrl), able to incorporate exogenous cyclic AMP.
Characterization of the aggregated states of glycogen phosphorylases by gel electrophoresis
Biochemistry, 1969
The disc gel electrophoretic method developed previously for the characterization of size and/or charge differences in proteins has been applied t o glycogen phosphorylase. We have concluded that phosphorylases a and b have the same molecular size but different net charges under the conditions of electrophoresis employed. The enzymes are both dimers with molecular weights of 170,00~180,000 g/mole at p H 8.5, 35", and a protein concentration of 0.5 mg/ml. Phosphorylase dimer can be dissociated to monomers using sodium dodecyl sulfate or 2 M urea a t 4". The binding of various ligands (adenosine monophosphate, glucose 6-phosphate, and dextrins) changes the relative mobility of phosphorylase. Plots of the logarithm of the relative mobility L'S. gel concentration in the presence of ligands yields lines parallel to the line obtained with phosphorylase alone. This
Biochemical Journal, 1975
1. In the isolated perfused rat liver, increasing glucose concentration from 5.5 to 55 mm in the perfusion medium caused a sequential inactivation of glycogen phosphorylase and activation of glycogen synthetase. The latter change was preceded by a lag period which corresponded to the time required to inactivate the major part of the phosphorylase. 2. The same sequence of events was observed in isolated rat hepatocytes incubated at 37°C. In this preparation, the rate of phosphorylase inactivation was greatly increased by increasing the concentration of glucose and/or of K+ ions in the external medium. The same agents also caused the activation of glycogen synthetase, but this effect was secondary to the inactivation of phosphorylase. 3. In both types of preparations, the rate of synthetase activation was modulated by the residual amount of phosphorylase a that remained after the initial phase of rapid inactivation and was independent of glucose concentration. 4. In isolated hepatocyt...
Journal of Biological Chemistry, 2001
We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mM glucose and by 2-fold at 20 mM glucose with a decrease in the concentration of glucose causing half-maximal rate (S 0.5 ) from 26 to 19 mM. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.