On the mechanism of hepatic glycogenolysis induced by anoxia or cyanide

Addition of glucagon to isolated hepatocytes increased glycogenolysis and phosphorylase a in a proportional manner. KCN caused slightly more glycogenolysis at considerably lower levels of phosphorylase a; the discrepancy was most pronounced after pretreatment of the hepatocytes with EGTA. When incubated with tagatose, the hepatocytes accumulated tagatose 1-phosphate, a presumed inhibitor of phosphorylase a. In these conditions the glucagon-induced glycogenolysis was blocked, but the glycogen loss caused by KCN or anoxia was not affected. Cyanide and anoxia may allow phosphorylase b and a to become equally active, or they may trigger a non-phosphorolytic glycogenolysis.     

Muscle glycogenolysis. Regulation of the cyclic interconversion of phosphorylase a and phosphorylase b

Regulation of glycogenolysis in skeletal muscle is dependent on a network of interacting enzymes and effectors that determine the relative activity of the enzyme phosphorylase. That enzyme is activated by phosphorylase kinase and inactivated by protein phosphatase-1 in a cyclic process of covalent modification. We present evidence that the cyclic interconversion is subject to zero-order ultrasensitivity, and the effect is responsible for the "flash" activation of phosphorylase by Ca2+ in the presence of glycogen. The zero-order effect is observable either by varying the amounts of kinase and phosphatase or by modifying the ratio of their activities by a physiological effector, protein phosphatase inhibitor-2. The sensitivity of the system is enhanced in the presence of the phosphorylase limit dextrin of glycogen which lowers the Km of phosphorylase kinase for phosphorylase. The in vitro experimental results are examined in terms of physiological conditions in muscle, and it is shown that zero-order ultrasensitivity would be more pronounced under the highly compartmentalized conditions found in that tissue. The sensitivity of this system to effector changes is much greater than that found for allosteric enzymes. Furthermore, the sensitivity enhancement increases more rapidly than energy consumption (ATP) as the phosphorylase concentration increases. Energy effectiveness is shown to be a possible evolutionary factor in favor of the development of zero-order ultrasensitivity in compartmentalized systems.     

Control of platelet glycogenolysis; Activation of phosphorylase kinase by calcium.

An apparent enigma during platelet aggregation is that increased glycogenolysis occurs despite a fall in cyclic AMP levels; Activation by a classical cascade is therefore unlikely, and an alternative stimulus for phosphorylase a formation was sought. It was found that low levels of Ca-2+ markedly activate phosphorylase b kinase from human platelets, with a Ka of 0i muM Ca-2+, which is similar to that for the skeletal muscle enzyme; The kinase activity is unstable, and on enzyme ageing is a 50% loss in activity with the Ka decreasing to 0.33 muM Ca-2+. In unstilulated platelets, phosphorylase a was 13.3% of toal measured activity, and glycogen synthetase I was 32.3%. Aggregation induced by ADP did not change the percentage of I synthetase, while increasing that for phosphorylase a. Dibutyryl cyclic AMP did, as expected, increase the percentage of both phosphorylated enzymes; These findings suggest that the natural activator of platelet glycogenolysis during aggregation is Ca-2+, which directly stimulates phosphorylase b kinase without altering glycogen synthetase activity. The cyclic AMP-dependent protein kinase does not appear to be involved;     

Cyclic AMP-mediated activation of hepatic glycogenolysis by fructose.

Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continuous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3', 5'-monophosphate (cyclic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results demonstrate that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.     

Regulation of immune-aggregate-stimulated hepatic glycogenolysis and vasoconstriction by vicinal dithiols.

Evidence suggesting that vicinal dithiols regulate immune-aggregate-induced vasoconstriction and glycogenolysis in the perfused rat liver was obtained. Phenylarsine oxide (PhAsO) and other tervalent organic arsenicals inhibited in a dose-dependent manner hepatic glycogenolysis, vasoconstriction, Ca2+ mobilization and the stimulated O2 consumption caused by immune-aggregate infusion. Polar tervalent and quinquivalent arsenicals were less effective than hydrophobic arsenicals. Prior infusion of Fc- but not Fab-fragments of IgG prevented partially immune-aggregate-stimulated hepatic metabolism, suggesting that immune aggregates elicit hepatic metabolic responses through Fc gamma receptors. The inhibitory action of PhAsO on immune-aggregate-stimulated hepatic glycogenolysis was unique; inhibition of glycogenolysis was not observed when phenylephrine, isoprenaline or glucagon was used as a stimulant. Although PhAsO might be expected to sequester cellular thiols, no significant change in the oxidation-reduction state of the major cellular thiol, glutathione, was found during PhAsO infusion. In addition, PhAsO exerted its effects without producing changes in hepatic adenine nucleotides and cyclic AMP. Evidence suggesting the involvement of vicinal dithiols was obtained through thiol-competition experiments using mono- and di-thiols. PhAsO inhibition of IgG-aggregate-stimulated hepatic vasoconstriction and glycogenolysis was reversed significantly by infusion of 2,3-dimercaptopropan-1-ol at 3-fold molar excess, whereas 2-mercaptoethanol at 40-fold molar excess was ineffective. The results of the present study provide evidence documenting the participation of vicinal dithiols during the coupling of hepatic immune-aggregate clearance by Kupffer cells with vasoconstriction of the hepatic vasculature (e.g. endothelial cells) and glycogenolysis (e.g. parenchymal cells).     

Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesis.

Multiple signalling pathways are involved in the mechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase-3 (GSK-3) and an allosteric inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a, to determine the relative contributions of inactivation of GSK-3 and dephosphorylation of phosphorylase a as alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes. GSK-3 inhibitors (SB-216763 and Li+) caused a greater activation of glycogen synthase than insulin (90% vs. 40%) but a smaller stimulation of glycogen synthesis (30% vs. 150%). The contribution of GSK-3 inactivation to insulin stimulation of glycogen synthesis was estimated to be less than 20%. Dephosphorylation of phosphorylase a with CP-91149 caused activation of glycogen synthase and translocation of the protein from a soluble to a particulate fraction and mimicked the stimulation of glycogen synthesis by insulin. The stimulation of glycogen synthesis by phosphorylase inactivation cannot be explained by either inhibition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translocation of synthase. Titrations with the phosphorylase inactivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phosphorylase a. We conclude that a signalling pathway involving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Selective inactivation of phosphorylase can mimic insulin stimulation of hepatic glycogen synthesis.     

The hepatic PP1 glycogen-targeting subunit interaction with phosphorylase a can be blocked by C-terminal tyrosine deletion or an indole drug

The inhibition of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) by glycogen phosphorylase a prevents the dephosphorylation and activation of glycogen synthase, suppressing glycogen synthesis when glycogenolysis is activated. Here, we show that a peptide ((280)LGPYY(284)) comprising the last five amino acids of G(L) retains high-affinity interaction with phosphorylase a and that the two tyrosines play crucial roles. Tyr284 deletion abolishes binding of phosphorylase a to G(L) and replacement by phenylalanine is insufficient to restore high-affinity binding. We show that a phosphorylase inhibitor blocks the interaction of phosphorylase a with the G(L) C-terminus, suggesting that the latter interaction could be targeted to develop an anti-diabetic drug.     

Asn112 in Plasmodium falciparum glutathione S-transferase is essential for induced reversible tetramerization by phosphate or pyrophosphate

The glutathione S-transferase from Plasmodium falciparum presents distinct features which are absent from mammalian GST isoenzyme counterparts. Most apparent among these are the ability to tetramerize and the presence of a flexible loop. The loop, situated between the 113–119 residues, has been reported necessary for the tetramerization process. In this article, we report that a residue outside of this loop, Asn112, is a key to the process — to the point where the single Asn112Leu mutation prevents tetramerization altogether. We propose that a structural pattern involving the interaction of the Asn112 and Lys117 residues from two neighboring subunits plays a role in keeping the tetramer structure stable. We also report that, for the tetramerization of the wild-type PfGST to occur, phosphate or pyrophosphate anions must be present. In other words, tetramerization is a phosphate- or pyrophosphate-induced process. Furthermore, the presence of magnesium reinforces this induction. We present experimental evidence for these claims as well as a preliminary calorimetric and kinetic study of the dimeric Asn112Leu PfGST mutant. We also propose a putative binding site for phosphate or pyrophosphate anions through a comparative structural analysis of PfGST and pyrophosphatases from several organisms. Our results highlight the differences between PfGST and the human isoenzymes, which make the parasite enzyme a suitable antimalarial target.     

Malonaldehyde formation in intact platelets is catalysed by thromboxane synthase.

Imidazole and compound L8027 (selective inhibitors of thromboxane synthase) produced parallel inhibition of malonaldehyde and thromboxane B2 secretion induced by collagen or thrombin in gel-filtered suspensions of human platelets. Comparing the effects of these inhibitors and aspirin on secretion of granule constituents indicated that platelet degranulation depends mainly on thromboxane production; prostaglandin endoperoxides contributed little.