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PubMed1983

Effects of glucose on phosphorylase and glycogen synthase in hepatocytes from diabetic rats.

The effects of glucose on phosphorylase and glycogen synthase were investigated in hepatocytes isolated from acutely (40 h) and chronically (90 h) alloxan-diabetic rats. The glucose-induced inactivation of phosphorylase proceeded normally in all conditions. The ensuing activation of glycogen synthase was slightly blunted in acute diabetes, but became virtually absent in 72 h diabetes of similar severity. In hepatocytes from rats with various degrees of chronic diabetes, the maximal activation of glycogen synthase (at 60 mM-glucose) was inversely correlated with the plasma glucose concentration.

Animals↗
PubMed1983

The denaturation of rabbit muscle phosphorylase b by guanidinium chloride.

The denaturation of phosphorylase b by guanidinium chloride (GdnHCl) was studied. The enzyme is unusually sensitive to the denaturing agent, being more than 50% inactivated after incubation for 15 min in 0.1 M-GdnHCl. Full activity can be regained on dilution of the GdnHCl to 0.01 M, provided that the initial concentration of GdnHCl is less than 0.5 M. Studies of protein fluorescence, thiol-group reactivity, circular dichroism and absorption spectroscopy indicate that phosphorylase b undergoes slow structural changes in the range of GdnHCl concentrations from 0.5 to 0.8 M. The enzyme retains considerable folded structure even after 15 min incubation in 1 M-GdnHCl, but is rapidly and completely unfolded in 3 M-GdnHCl.

Animals↗
PubMed1986

Study of the hydrolysis and ionization constants of Schiff base from pyridoxal 5'-phosphate and n-hexylamine in partially aqueous solvents. An application to phosphorylase b.

Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate.

Amines↗
PubMed1995

Mechanism of regulation in yeast glycogen phosphorylase.

The mechanism of yeast glycogen phosphorylase activation by covalent phosphorylation involves structural elements distinct from the mammalian homologs. To understand the role of the amino-terminal 39-residue extension in the phosphorylation control mechanism, mutants with 22 and 42 amino-terminal residues removed were expressed in Escherichia coli, and their properties were compared with the wild-type (WT) enzyme. The unphosphorylated WT enzyme had a specific activity of 0.1 unit/mg and was not activated significantly by the substrate, glucose 1-phosphate. Phosphorylation by protein kinase resulted in a 1300-fold activation. Glucose 6-phosphate inhibited the unphosphorylated enzyme more effectively than the phosphorylated form, and inhibition of the latter was cooperative. Glucose was a poor inhibitor for both the unphosphorylated and phosphorylated WT enzyme with Ki > 300 mM. The rate of phosphorylation by protein kinase depended on substrates and interactions of the amino terminus. Maltoheptaose increased the rate of phosphorylation of the WT enzyme by yeast phosphorylase kinase 5-fold. The 22-residue deletion mutant (Nd22) had overall kinetic properties similar to the WT enzyme, except that Nd22 was a better substrate for the protein kinase and the rate of phosphorylation was unaffected by maltoheptaose. The 42-residue deletion mutant (Nd42), which lacks the phosphorylation site, was measurably active, although much less active than phosphorylated WT. Sedimentation equilibrium analysis indicated that the WT, Nd22, and Nd42 exist as tetramer, partially dissociated tetramer, and dimer, respectively. Phosphorylation of the WT and Nd22 converted both to dimer. The results indicated that the amino terminus affects quaternary structure and mediates activity regulation through conformational transition.

Base Sequence↗
PubMed2004

Characterization of Escherichia coli uridine phosphorylase by single-site mutagenesis.

The Escherichia coli udp gene encodes uridine phosphorylase (UP), which catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. The X-ray structure of E. coli UP resolved by two different groups produced conflicting results. In order to cast some light on the E. coli UP catalytic site, we mutagenized several residues in UP and measured by RP-HPLC the phosphorolytic activity of the mutant UP proteins in vitro. Mutations Thr94Ala, Phe162Ala, and Tyr195Gly caused a drastic decrease in UP activity. These three residues were suggested to be involved in the nucleoside binding site. However, surprisingly, Tyr195Ala caused a relative increase in enzymatic activity. Both Met197Ala and Met197Ser conserved low activity, suggesting a minor role for this residue in the UP active site. Glu196Ala completely lost UP activity, whereas the more conservative Glu196Asp mutation was still partially active, confirming the importance of maintaining the correct charge in the surroundings of this position. Glu198 was mutated to either Gly, Asp and Gln. All three substitutions caused complete loss of enzymatic activity suggesting an important role of Glu198 both in ribose binding and in interaction with phosphate ions. Arg30Ala and Arg91Ala eliminated UP activity, whereas Arg30Lys and Arg91Lys presented a very low activity, confirming that these residues might interact with and stabilize the phosphate ions. Ile69Ala did not decrease UP activity, whereas His8Ala lowered the activity to about 20%. Both amino acids were suggested to take part in subunit interactions. Our results confirm the structural similarity between E. coli UP and E. coli purine nucleoside phosphorylase (PNP).

Amino Acid Sequence↗
PubMed1989

Domain separation in the activation of glycogen phosphorylase a.

The crystal structure of glycogen phosphorylase a complexed with its substrates, orthophosphate and maltopentaose, has been determined and refined at a resolution of 2.8 angstroms. With oligosaccaride bound at the glycogen storage site, the phosphate ion binds at the catalytic site and causes the regulatory and catalytic domains to separate with the loss of stabilizing interactions between them. Homotropic cooperativity between the active sites of the allosteric dimer results from rearrangements in isologous contacts between symmetry-related helices in the subunit interface. The conformational changes in the core of the interface are correlated with those observed on covalent activation by phosphorylation at Ser14 (phosphorylase b----a).

Allosteric Site↗
PubMed1986

Adult muscle phosphorylase "b" kinase deficiency.

A 35-year-old man had severe exercise intolerance and cramps. Venous blood lactate did not rise after ischemic exercise, and electromyographically silent contracture of hand muscles appeared. Histochemistry and electronmicroscopy of a muscle biopsy revealed subsarcolemmal and intermyofibrillar accumulation of glycogen. Biochemical studies showed moderately increased amount of glycogen. Total phosphorylase activity was normal, but the active form "a" was 27% of normal. Phosphorylase kinase activity was 12% of the normal value and was normal in leukocytes and erythrocytes.

Adult↗
PubMed1991

Purifications and properties of orotidine-phosphorolyzing enzyme and purine nucleoside phosphorylase from Erwinia carotovora AJ 2992.

An orotidine-phosphorolyzing enzyme and a purine nucleoside phosphorylase (PNPase) of Erwinia carotovora AJ 2992, which is a potent producer of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), an antiviral agent, from orotidine and 1,2,4-triazole-3-carboxamide (TCA), were purified 23-fold and 103-fold, respectively. At each purification step, the orotidine-phosphorolyzing enzyme was always co-purified with an uridine phosphorylase (UPase) and its activity could not be separated from that of the UPase after it showed as a single band on SDS-polyacrylamide gel electrophoresis. These results suggest that this enzyme may be identical with UPase. The purified enzyme had a molecular weight of 68,000 +/- 2,000, and seemed to be a dimer. The optimal temperatures and pH values were 60 degrees C and 6.0 for orotidine phosphorolysis, and 70 degrees C and 7.0 for uridine phosphorolysis. The Michaelis constants for uridine and orotidine were 0.75 mM and 10.87 mM, respectively, at 40 degrees C. The PNPase of E. carotovora AJ 2992 had a molecular weight of 58,000 +/- 2,000 and seemed to be a dimer. The Michaelis constants for inosine and guanosine were 1.92 mM and 1.85 mM, respectively, at 40 degrees C. The PNPase was completely inactivated by p-chloromercuribenzoate.

Amino Acid Sequence↗
PubMed1977

Regulation of phosphorylase-phosphatase from skeletal muscle by phosphorylation of a regulator protein.

The inhibitory effect of a heat-stable regulator protein from skeletal muscle on the activity of phosphorylase-phosphatase (EC 3.1.3.17) was studied. The regulator protein was shown to be both phosphorylated and dephosphorylated in vivo as well as in vitro. The incorporation of phosphate into the regulator protein increased, while dephosphorylation decreased the ability of the protein to inhibit phosphatase activity. Our results suggest that the reversible phosphorylation of the regulator protein plays an essential role in the regulation of phosphorylase-phosphatase activity.

Animals↗
PubMed1979

The stress induced increase of liver phosphorylase A and cyclic AMP in adrenomedullectomized and adrenalectomized rats.

In adult male rats injected with Pentobarbital, 50 mg.kg-1 i.p. and subjected immediately after administration of the anaesthetic to 400 revolutions (lasting of 6 min and 40 s) in rotating Noble-Collip drums the activity of the active form of hepatic phosphorylase was increased at time 0 after injury without respect to previous adrenomedullectomy or adrenalectomy (7 weeks or 10 days before, respectively). Completeness of surgery was checked by plasma catecholamines which were essentially near zero. The level of liver cAMP was increased in intact and adrenomedullectomized animals at time zero. 90 min after injury a recovery of enzyme activity towards basal levels was observed in contrast to cAMP which was increased in all the three groups. A net glycogenolytic response was found in the injured animals irrespective of previous surgery. It is concluded that for the stress induced activation of hepatic phosphorylase the presence of adrenal cortical and medullary tissue is not always indispensable.

Adrenal Medulla↗
PubMed1989

Thermodynamic analysis of the activation of glycogen phosphorylase b over a range of temperatures.

Equilibrium dialysis and isothermal microcalorimetry experiments have been carried out to characterize the thermodynamics of the binding of AMP to glycogen phosphorylase b (EC 2.4.1.1) at pH 6.9 over the temperature range of 25-35 degrees C. Thermal titrations were performed at each temperature in various buffer systems, which have afforded the calculation of the number of protons exchanged when the AMP binds to each site in the protein. Thermodynamic parameters were obtained for the binding of AMP to the two nucleotide and the two inhibitor sites of the dimeric enzyme. The former show positive cooperativity while the latter behave as independent binding sites. A positive delta Cp value was obtained for the AMP binding to the two N sites (1.3 and 1.4 kJ K-1 mol-1), while the delta Cp was negative for the binding to the I sites (-1.9 kJ K-1 mol-1). The application of Sturtevant's method to our data (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240) and their comparison with a similar analysis undertaken with phosphorylase a (Mateo, P. L., González, J. F., Barón, C., Lopez-Mayorga, O., and Cortijo, M. (1986) J. Biol. Chem. 261, 17067-17072) has opened the way to some understanding of the thermodynamics of the allosteric transition in the protein.

Animals↗
PubMed1986

Inhibition of muscle glycogen phosphorylase b by vitamin B2 and its coenzyme forms.

Kinetic studies have demonstrated that vitamin B2 and its coenzyme forms FMN and FAD are potent inhibitors of glycogen phosphorylase b from rabbit skeletal muscle. The inhibition of the enzyme by flavins has a co-operative character (Hill coefficients exceed unity). Glycogen phosphorylase b bound to FMN or FAD does not reveal catalytic activity, whereas the enzyme bound to riboflavin retains about 16% of the initial catalytic activity.

Animals↗
PubMed1986

Lanthanide ions and Cd2+ are able to substitute for Ca2+ in regulating phosphorylase kinase.

Trivalent lanthanide ions and Cd2+ were found to mimic effectively the stimulatory action of Ca2+ on rabbit muscle phosphorylase kinase. In the range of concentrations tested, Cd2+ and lanthanides (Tb3+, Gd3+, Pr3+, Ce3+) could substitute for Ca2+ in activating the enzyme to about 60% and 70% respectively of the maximal level seen with Ca2+, at pH 8.2. The effect induced by Cd2+ was biphasic (stimulation followed by inhibition with increasing metal cation concentration). Similar results were obtained at pH 6.8. Cd2+ and Tb3+ were also able to replace Ca2+ required for the stimulation of phosphorylase kinase activity at pH 8.2 by exogenous calmodulin. Maximal stimulation induced by calmodulin in presence of Cd2+ was significantly higher than that in presence of Ca2+ or Tb3+.

Animals↗
PubMed1987

Glycogen phosphorylase in Dictyostelium. Developmental regulation of two forms and their physical and kinetic properties.

A key step in the cellular differentiation of Dictyostelium is the degradation of glycogen to provide the precursors for synthesis of the structural end products of development. We have found that the enzyme that initiates this degradative pathway, glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1), is developmentally regulated and exists as two forms. During the time course of development, a previously undescribed activity, the "b" form, decreases, whereas that of the "a" form increases. The b form is inactive unless 5'-AMP is included in the reaction mixture. The mechanism of activation by 5'-AMP is by a 40-fold increase in the affinity of the phosphorylase for its substrates. Both forms were purified to homogeneity. They have identical subunit molecular weights of 90,000 and both exist as a dimer under non-denaturing conditions. The two forms are also identical with respect to salt inhibition, optimum temperature for activity, and pH optimum. They differ in their elution from DE52-cellulose, affinity constants, thermal stability, affinity for 5'-AMP-Sepharose, and their peptide maps. Attempts to demonstrate interconversion of the two activities by a kinase-directed phosphorylation have been unsuccessful. We report here on the existence, the developmental regulation, the purification to homogeneity, and some of the physical and kinetic properties of both the 5'-AMP-dependent and -independent forms of the enzyme.

Dictyostelium↗
PubMed1985

Glycogen content and phosphorylase activities in deciduous dental pulp. Study on porcine teeth during root formation, fully formed root and root resorption phases.

Glycogen content and glycogen phosphorylases (a and total) were measured in dental pulps from pigs sacrificed at 20, 40, 50, 70, 90, 120, 180, 270 and 300 days of age. The following conclusions were drawn: (a) pulp glycogen content was higher during root formation than during root resorption; (b) the phosphorylase activities (a and total), were higher during root resorption than in the other phases; (c) pulps from different teeth, when in the same developmental stage, possessed a similar glycogen metabolism, as revealed by glycogen content and enzyme activity determinations.

Aging↗
PubMed1985

[Interaction of muscle glycogen phosphorylase B with flavin-adenine dinucleotide and its analogs].

The inhibition of rabbit skeletal muscle glycogen phosphorylase b by FAD and its analogues with substitutes in the position 8 has been studied. The value of half-saturation, [I]0,5, for inhibitors increases in the following order: FAD (44 microM), 8 alpha-hydroxy-FAD (60 microM), 8-dimethylamino (nor)-FAD (69 microM), 8 alpha-(N-acetyl-L-cystein-S-yl)-FAD (106 microM). From the comparison of these values with those obtained earlier for FMN analogues, it follows that in the case of FAD the half-saturation value is less sensitive to modification of the position 8 in the flavin isoalloxazine ring. The existence of the glycogen phosphorylase b FAD-complex has been proved by the spectrophotometry and sedimentation methods. The positions of maxima of optical absorption of the enzyme-bound FAD in the 300-500 nm region are identical with corresponding positions for FMN. FAD has been shown to hinder the AMP-induced transition of dimeric form of the enzyme to tetrameric one.

Animals↗
PubMed1979

[Effect of polyethylene glycol on association of muscle phosphorylase B].

It has been shown that a neutral polymer poly(ethyleneglycol) with molecular weight of 4000 and more shifts the equilibrium between the dimer and tetramer forms of phosphorylase B from rabbit skeletal muscles in the direction of formation of species with higher molecular weights. Poly(ethyleneglycol) with molecular weight below 600 has no effect on the association process. Phosphorylase B is precipitated in the form of crystals in the presence of poly(ethyleneglycol) mol. weight 40,000), when the concentration of the latter is higher than 50 mg/ml. AMP and sulfate ions are required for the crystallization process.

Chemical Phenomena↗
PubMedDate not supplied

[Binding of muscle phosphorylase B by glycogen].

The binding of phosphorylase B with glycogen has been studied by means of analytical ultracentrifuge with absorption optics and a photoelectric scanner. Adsorbtion capacity for pig liver glycogen with respect to phosphorylase B (aM) and microscopic dissociation constant of enzyme -- glycogen complex (K) have been determined (aM = 3.64 . 10(-6) moles of bound enzyme per 1 g of glycogen: K = 2.6 . 10(-7) M at 12.7 degrees, pH 6.8). For oyster glycogen the value of aM is equal to 3.92 . 10(-6) moles of the bound protein per 1g of glycogen and K = 6.8 . 10(-7) M. The method of determination of microscopic Michaelis constant with respect to glycogen using known value of aM has been demonstrated.

Animals↗