Integrated effects of multiple modulators on human liver glycogen phosphorylase a (original) (raw)
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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.
Allosteric Regulation of Liver Phosphorylasea: Revisited under Approximated Physiological Conditions
Archives of Biochemistry and Biophysics, 1996
ity compared to the activity without any effectors. In addition, changes in glucose concentration did not af-Phosphorylase removes glucosyl units from the terfect activity. K glutamine modestly stimulated activminal branches of glycogen through phosphorolysis, ity. Numerous other metabolites were tested and were forming glucose-1-P. It is present in two interconvertwithout effect. The present data indicate that the ible forms, phosphorylase a and b. The a form is the known endogenous allosteric effectors cannot explain active form and is rate limiting in glycogen degradathe smaller than expected in vivo phosphorylase a action. The activities of phosphorylase a and of total tivity or the regulation of phosphorylase a activity. phosphorylase as conventionally measured exceed the ᭧ 1996 Academic Press, Inc.
Biochemical Journal, 1995
A series of glucose-analogue inhibitors of glycogen phosphorylase b (GPb) has been designed, synthesized and investigated in crystallographic binding and kinetic studies. The aim is to produce a compound that may exert more effective control over glycogen metabolism than the parent glucose molecule and which could alleviate hyperglycaemia in Type-II diabetes. N-Acetyl-beta-D-glucopyranosylamine (1-GlcNAc) has a Ki for muscle GPb in crude extracts of 30 microM, 367-fold lower than that of beta-D-glucose [Board, Hadwen and Johnson (1995) Eur. J. Biochem. 228, 753-761]. In the current work, the effects of 1-GlcNAc on the activation states of GP and glycogen synthase (GS) in cell-free preparations and in isolated hepatocytes are reported. In gel-filtered extracts of liver, which lack ATP for kinase activity, 1-GlcNAc produced a rapid and time-dependent inactivation of GP with a subsequent activation of GS. Effects of 1-GlcNAc on both enzymes were stronger than those of glucose, with 0.8...
Diabetes, 2000
The racemic prodrug BAY R3401 suppresses hepatic glycogenolysis. BAY W1807, the active metabolite of BAY R3401, inhibits muscle glycogen phosphorylase a and b. We investigated whether BAY R3401 reduces hepatic glycogenolysis by allosteric inhibition or by phosphatasecatalyzed inactivation of phosphorylase. In gel-filtered liver extracts, racemic BAY U6751 (containing active BAY W1807) was tested for inhibition of phosphorylase in the glycogenolytic (in which only phosphorylase a is active) and glycogen-synthetic (for the evaluation of a:b ratios) directions. Phosphorylase inactivation by endogenous phosphatase was also studied. In liver extracts, BAY U6751 (0.9-36 µmol/l) inhibited glycogen synthesis by phosphorylase b (notwithstanding the inclusion of AMP), but not by phosphorylase a. Inhibition of phosphorylase-a-catalyzed glycogenolysis was partially relieved by AMP (500 µmol/l). BAY U6751 facilitated phosphorylase-a dephosphorylation. Isolated hepatocytes and perfused livers were tested for BAY R3401-induced changes in phosphorylase-a:b ratios and glycogenolytic output. Though ineffective in extracts, BAY R3401 (0.25 µmol/l-0.5 mmol/l) promoted phosphorylase-a dephosphorylation in hepatocytes. In perfused livers exposed to dibutyryl cAMP (100 µmol/l) for maximal activation of phosphorylase, BAY R3401 (125 µmol/l) inactivated phosphorylase by 63% but glucose output dropped by 83%. Inhibition of glycogenolysis suppressed glucose-6-phosphate (G6P) levels. Activation of glycogen synthase after phosphorylase inactivation depended on the maintenance of G6P levels by supplementing glucose (50 mmol/l). We conclude that the
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 calorimetric study of the binding of AMP to liver glycogen phosphorylase b
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1996
The energetics of the interaction between liver glycogen phosphorylase b and the adenosine 5'-monophosphate (AMP) have been studied by equilibrium dialysis and isothermal titration calorimetry (ITC) at 25°C. A concomitant net release of protons with AMP to phosphorylase binding was detected carrying out calorimetric experiments in three buffers having different heats of ionization at 25°C. Four binding sites were found for AMP in the dimeric enzyme, which would correspond to the activator and the inhibitor sites identified in the muscle isozyme. The affinity of AMP for these four sites is similar. Thus, the binding of AMP to the activator sites seems to be non-cooperative and it does not perform the conformational change necessary to activate the enzyme. Moreover, the inhibitor sites are occupied almost in the same extension that the activator sites, which would impair any activation of the enzyme.