Synthesis and kinetic evaluation of 4-deoxymaltopentaose and 4-deoxymaltohexaose as inhibitors of muscle and potato alpha-glucan phosphorylases. (1/602)

alpha-Glucan phosphorylases degrade linear or branched oligosaccharides via a glycosyl transfer reaction, occurring with retention of configuration, to generate alpha-glucose-1-phosphate (G1P). We report here the chemoenzymic synthesis of two incompetent oligosaccharide substrate analogues, 4-deoxymaltohexaose (4DG6) and 4-deoxymaltopentaose (4DG5), for use in probing this mechanism. A kinetic analysis of the interactions of 4DG5 and 4DG6 with both muscle and potato phosphorylases was completed to provide insight into the nature of the binding mode of oligosaccharide to phosphorylase. The 4-deoxy-oligosaccharides bind competitively with maltopentaose and non-competitively with respect to orthophosphate or G1P in each case, indicating binding in the oligosaccharide binding site. Further, 4DG5 and 4DG6 were found to bind to potato and muscle phosphorylases some 10-40-fold tighter than does maltopentaose. Similar increases in affinity as a consequence of 4-deoxygenation were observed previously for the binding of polymeric glycogen analogues to rabbit muscle phosphorylase [Withers (1990) Carbohydr. Res. 196, 61-73].  (+info)

Stimulation of phosphorylase kinase autophosphorylation by peptide analogs of phosphorylase. (2/602)

Autoactivation of phosphorylase kinase in the presence of substrates has been studied to determine the cause of the hysteresis, or lag, in the phosphorylase kinase reaction. Peptide analogs corresponding to the convertible serine region of phosphorylase have been used as low molecular weight alternative substrates. Autophosphorylation of the kinase molecule was measured under conditions that favored autoactivation. Phosphorylase b and a tetradecapeptide, which was found to be a good model of phosphorylase, stimulated autoactivation by 86- and 37-fold, respectively. The tetradecapeptide also stimulated autophosphorylation of subunits A and B of the kinase molecule. This increased autophosphorylation coincided with an increased ability to convert phosphorylase. This finding supports the hypothesis that autophosphorylation is responsible for the lag in the phosphorylase kinase reaction. No evidence was obtained to suggest that the lag could be due to dissociation of the kinase. The stoichiometry of phosphate incorporation into phosphorylase kinase subunits by autophosphorylation was much greater than that reported to occur by protein kinase phosphorylation. Multiple phosphorylation sites in subunit A accounted for most of the phosphate incorporation during autophosphorylation. Saturating levels of hexa- and octapeptide analogs also caused stimulation of autophosphorylation. Possible mechanisms and experimental implications of substrate-stimulated autophosphorylation are discussed. Consideration also is given to the possible role of effectors in autophosphorylation in vivo.  (+info)

Human liver glycogen phosphorylase. Kinetic properties and assay in biopsy specimens. (3/602)

1. The two forms of glycogen phosphorylase were purified from human liver, and some kinetic properties were examined in the direction of glycogen synthesis. The b form has a limited catalytic capacity, resembling that of the rabbit liver enzyme. It is characterized by a low affinity for glucose 1-phosphate, which is unaffected by AMP, and a low V, which becomes equal to that of the a form in the presence of the nucleotide. Lyotropic anions stimulate phosphorylase b and inhibit phosphorylase a by modifying the affinity for glucose 1-phosphate. Both enzyme forms are easily saturated with glycogen. 2. These kinetic properties have allowed us to design a simple assay method for total (a + b) phosphorylase in human liver. It requires only 0.5 mg of tissue, and its average efficiency is 90% when the enzyme is predominantly in the b form. 3. The assay of total phosphorylase allows the unequivocal diagnosis of hepatic glycogen-storage disease caused by phosphorylase deficiency. One patient with a complete deficiency is reported. 4. The assay of human liver phosphorylase a is based on the preferential inhibition of the b form by caffeine. The a form displays the same activity when measured by either of the two assays.  (+info)

Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure. (4/602)

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.  (+info)

Glycogen depletion rather than glucose 6-P increments controls early glycogen recovery in human cultured muscle. (5/602)

In glycogen-containing muscle, glycogenesis appears to be controlled by glucose 6-phosphate (6-P) provision, but after glycogen depletion, an autoinhibitory control of glycogen could be a determinant. We analyzed in cultured human muscle the contribution of glycogen depletion versus glucose 6-P in the control of glycogen recovery. Acute deglycogenation was achieved by engineering cells to overexpress glycogen phosphorylase (GP). Cells treated with AdCMV-MGP adenovirus to express 10 times higher active GP showed unaltered glycogen relative to controls at 25 mM glucose, but responded to 6-h glucose deprivation with more extensive glycogen depletion. Glycogen synthase (GS) activity ratio was double in glucose-deprived AdCMV-MGP cells compared with controls, despite identical glucose 6-P. The GS activation peak (30 min) induced by glucose reincubation dose dependently correlated with glucose 6-P concentration, which reached similar steady-state levels in both cell types. GS activation was significantly blunted in AdCMV-MGP cells, whereas it strongly correlated, with an inverse relationship, with glycogen content. An initial (0-1 h) rapid insulin-independent glycogen resynthesis was observed only in AdCMV-MGP cells, which progressed up to glycogen levels approximately 150 micrograms glucose/mg protein; control cells, which did not deplete glycogen below this concentration, showed a 1-h lag time for recovery. In summary, acute deglycogenation, as achieved by GP overexpression, caused the activation of GS, which inversely correlated with glycogen replenishment independent of glucose 6-P. During glycogen recovery, the activation promoted by acute deglycogenation rendered GS effective for controlling glycogenesis, whereas the transient activation of GS induced by the glucose 6-P rise had no impact on the resynthesis rate. We conclude that the early insulin-independent glycogen resynthesis is dependent on the activation of GS due to GP-mediated exhaustion of glycogen rather than glucose 6-P provision.  (+info)

McArdle's disease. The unsolved mystery of the reappearing enzyme. (6/602)

We assessed the frequency of muscle fibers showing histochemical phosphorylase activity in 27 muscle biopsies from 25 unrelated patients with McArdle's disease and studied by immunohistochemistry and in situ hybridization whether the muscle-specific isoform was expressed. Positive phosphorylase fibers were observed in 19% of our series of biopsies. We show that the enzyme isoform expressed in regenerating fibers differs according to the genotype of patients: the muscle-specific isoform is transcribed and translated in patients with none of the described mutations in at least one allele of the myophosphorylase gene, whereas it is neither transcribed nor translated in patients with identified mutations in both alleles.  (+info)

Influence of substrates on in vitro dephosphorylation of glycogen phosphorylase a by protein phosphatase-1. (7/602)

The kinetic theory of the substrate reaction during modification of enzyme activity has been applied to a study of the dephosphorylation of phosphorylase a by protein phosphatase-1 (ppase-1). On the basis of the kinetic equation of the substrate reaction in the presence of ppase-1, all the inactivation rate constants for the free enzyme and the enzyme-substrate(s) complexes have been determined. Binding of the allosteric substrate, glucose 1-phosphate, to one subunit of phosphorylase a protects completely against ppase-1 action on either the same subunit or the adjacent subunit, whereas binding of the non-allosteric substrate, glycogen, to one subunit protects this subunit partially, but has no effect on the modification on the neighbouring subunit. Analysis of the data suggests that the allosteric behaviour of phosphorylase a can be interpreted in terms of a modified concerted model. The present method also provides a novel approach for studying dephosphorylation reactions. Since the experimental conditions used resemble more closely the in vivo situation where the substrate is constantly being turned over while the enzyme is being modified, this new method would be particularly useful when the regulatory mechanism of the reversible phosphorylation reaction toward certain enzymes is being assessed.  (+info)

Sparing effect of leptin on liver glycogen stores in rats during the fed-to-fasted transition. (8/602)

The effect of moderate hyperleptinemia ( approximately 20 ng/ml) on liver and skeletal muscle glycogen metabolism was examined in Wistar rats. Animals were studied approximately 90 h after receiving recombinant adenoviruses encoding rat leptin (AdCMV-leptin) or beta-galactosidase (AdCMV-betaGal). Liver and skeletal muscle glycogen levels in the fed and fasted (18 h) states were similar in AdCMV-leptin- and AdCMV-betaGal-treated rats. However, after delivery of a glucose bolus, liver glycogen levels were significantly greater in AdCMV-leptin compared with AdCMV-betaGal rats (P < 0.05). To investigate the mechanism(s) of these differences, glycogen levels were measured immediately after the cessation of a 3- or 6-h glucose infusion or 3, 6, and 9 h after the cessation of a 6-h glucose infusion. Similar increases in liver and skeletal muscle glycogen occurred in hyperleptinemic and control rats in response to glucose infusions. However, 3 and 6 h after the cessation of a glucose infusion, liver glycogen levels were approximately twofold greater (P < 0.05) in AdCMV-leptin-treated compared with AdCMV-betaGal-treated animals. Skeletal muscle glycogen levels were similar in AdCMV-leptin-treated and AdCMV-betaGal-treated animals at the same time points. Glycogen phosphorylase, phosphodiesterase 3B, and glycogen synthase activities were unaltered by hyperleptinemia. We conclude that moderate increases in plasma leptin levels decrease liver glycogen degradation during the fed-to-fasted transition.  (+info)