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At least 973 records · Page 54Linked to original sources
PubMed1989

Isolation, characterization, and inactivation of the APA1 gene encoding yeast diadenosine 5',5'''-P1,P4-tetraphosphate phosphorylase.

The gene encoding diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase from yeast was isolated from a lambda gt11 library. The DNA sequence of the coding region was determined, and more than 90% of the deduced amino acid sequence was confirmed by peptide sequencing. The Ap4A phosphorylase gene (APA1) is unique in the yeast genome. Disruption experiments with this gene, first, supported the conclusion that, in vivo, Ap4A phosphorylase catabolizes the Ap4N nucleotides (where N is A, C, G, or U) and second, revealed the occurrence of a second Ap4A phosphorylase activity in yeast cells. Finally, evidence is provided that the APA1 gene product is responsible for most of the ADP sulfurylase activity in yeast extracts.

Amino Acid Sequence↗
PubMed1989

Stabilization of the 3' one-third of Escherichia coli ribosomal protein S20 mRNA in mutants lacking polynucleotide phosphorylase.

Mutations which largely inactivate polynucleotide phosphorylase and which render RNase II thermolabile exert two effects on the metabolism of the two nested mRNAs which encode ribosomal protein S20. (i) The lifetime of both mRNA species is extended 2.5-fold at 38 degrees C in a strain harboring both mutations. (ii) A relatively stable truncated fragment of these mRNAs accumulates to significant levels in strains lacking polynucleotide phosphorylase. The truncated RNA (Po RNA) is 147 to 148 residues long and is coterminal with the 3' ends of intact S20 mRNAs. Its 5' end appears to be generated by endonucleolytic cleavage to the 5' side of a G residue in the sequence AACCGAUC. The data are consistent with the hypothesis that S20 mRNAs can be degraded by alternative pathways. The normal pathway depends on functional polynucleotide phosphorylase and is concerted, since S20 mRNAs disappear without accumulation of detectable intermediates in the decay process. The slower alternative pathway is followed when polynucleotide phosphorylase is inactivated by mutation. This pathway is distinguished by segmental rather than concerted degradation of S20 mRNAs and involves at least one endonucleolytic cleavage. The 5' two-thirds of S20 mRNAs decays significantly more quickly than the 3' third in this latter mode of mRNA turnover.

Alleles↗
PubMed2002

Gene encoding a trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047.

A gene encoding a trehalose phosphorylase was cloned from Thermoanaerobacter brockii ATCC 35047. The gene encodes a polypeptide of 774 amino acid residues. The deduced amino acid sequence was homologous to bacterial maltose phosphorylases and a trehalose 6-phosphate phosphorylase catalyzing anomer-inverting reactions. On the other hand, no homology was found between the T. brockii enzyme and an anomer-retaining trehalose phosphorylase from Grifola frondosa.

Amino Acid Sequence↗
PubMed1985

Temporal relationships between isometric force, phosphorylase, and protein kinase activities in vascular smooth muscle.

Vascular smooth muscle contractility is tightly coupled to ATP production by intermediary metabolism. To elucidate mechanisms underlying coordination of metabolism and contractility we studied the time course of isometric force, and the activation of phosphorylase and cAMP-dependent protein kinases during stimulation of bovine coronary arterial strips with KCl. Isometric force reached a maximum after 10 min of exposure to 30 mM KCl (ED90) and was sustained throughout the subsequent 20-min period of contraction. In contrast, activation of phosphorylase was biphasic: enzymic activity reached a maximum (176 +/- 10% of control) after 3 min of contraction and then, though remaining above control, activity declined to a lower level (135 +/- 7% of control). However, no change occurred in the activity ratios for cAMP-dependent protein kinase assessed in either the presence (type II isozyme) or absence (type I isozyme) of 0.5 M NaCl. These data suggest that the activation of phosphorylase during K+-induced contraction is independent of the cAMP system. The biphasic activation of phosphorylase may reflect transient changes in the intracellular concentration of Ca2+ or the activation of a phosphatase(s) during the response.

Animals↗
PubMed1986

Diadenosine 5',5'''-P1, P4-tetraphosphate alpha, beta-phosphorylase from yeast supports nucleoside diphosphate-phosphate exchange.

Homogeneous diadenosine 5',5'''-P1,P4-tetraphosphate alpha, beta-phosphorylase (Ap4A-phosphorylase), the enzyme recently found in yeast (Guranowski, A., and Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547) catalyzes an exchange reaction between the beta-phosphate of nucleoside diphosphate (NDP) and orthophosphate from the medium (Pi). The common purine and pyrimidine ribonucleoside diphosphates as well as ADP analogs modified either in aglycone, sugar, or at the anhydride bond beta-position are substrates. The Km and rate values for the NDP-Pi exchange reaction were estimated at pH optima. These optima are 6.5 for UDP, 7.0 for ADP or CDP, and 8.0 for GDP. In the presence of 10 mM K2HPO4, 0.1 mM EDTA, and 100 mM Hepes/KOH (pH 7.0), the Km for ADP is 0.7 mM with a rate constant at saturating ADP of 96 s-1. The Km value for orthophosphate is 2 mM. In the NDP-Pi exchange reaction, phosphate can be substituted with arsenate and apparent arsenolysis of NDPs yields corresponding nucleoside monophosphates. The same pH optimum of 6.5 is found for arsenolysis of ADP, GDP, and CDP. Whereas the Ap4A phosphorylase sulfhydryl groups are essential for catalyzing the Ap4A phosphorolysis, the NDP-Pi exchange reactions, and the arsenolysis of NDPs, the divalent metal ions (Mg2+, Mn2+, Ca2+, Co2+, and Cd2+), which had been shown to be essential cofactors of the former reaction, are not required for the two latter ones. Used at concentrations which are optimum for Ap4A phosphorolysis, the cations (particularly Mg2+ and Cd2+) inhibit the NDP-Pi exchange and the arsenolysis of NDPs. Interestingly, the Ap4A phosphorylase exhibits higher specificity for adenosine 5'-phosphosulfate (APS) than for any other NDP tested. The V/Km ratio is almost 5-fold higher with APS than with ADP. However, in the presence of orthophosphate, the APS is irreversibly converted to ADP. Thus, the enzyme displays a property already attributed to ADP-sulfurylase (EC 2.7.7.5), (Grunberg-Manago, M., Del Campillo-Campbell, A., Dondon, L., and Michelson, A. M. (1966) Biochim. Biophys. Acta 123, 1-16; Nicholls, R. G. (1977) Biochem. J. 165, 149-155).

Adenosine Diphosphate↗
PubMed1987

Catalysis in the crystal: synchrotron radiation studies with glycogen phosphorylase b.

Direct observation of the progress of a catalysed reaction in crystals of glycogen phosphorylase b has been made possible through fast crystallographic data collection achieved at the Synchrotron Radiation source at Daresbury, UK. In the best experiments, data to 2.7 A resolution (some 108,300 measurements; 21,200 unique reflections) were measured in 25 min. In a series of time-resolved studies in which the control properties of the enzyme were exploited in order to slow down the reaction, the conversion of heptenitol to heptulose-2-phosphate, the phosphorylysis of maltoheptaose to yield glucose-1-phosphate and the oligosaccharide synthesis reaction involving maltotriose and glucose-1-phosphate have been monitored in the crystal. Changes in electron density in the difference Fourier maps are observed as the reaction proceeds not only at the catalytic site but also the allosteric and glycogen storage sites. Phosphorylase b is present in the crystals in the T state and under these conditions exhibits low affinity for both phosphate and oligosaccharide substrates. There are pronounced conformational changes associated with the formation and binding of the high-affinity dead-end product, heptulose-2-phosphate, which show that movement of an arginine residue, Arg 569, is critical for formation of the substrate-phosphate recognition site. The results are discussed with reference to proposals for the enzymic mechanism of phosphorylase. The feasibility for time-resolved studies on other systems and recent advances in this area utilizing Laue diffraction are also discussed.

Animals↗
PubMed1986

Properties of muscle phosphorylase b from a hagfish, Paramyxine atami.

Phosphorylase b was purified to homogeneity from the muscle of a hagfish (Paramyxine atami), as judged by electrophoretic and immunological criteria. The purified enzyme was partially but not fully activated by AMP, and its conversion into the a form resulted in a three-fold increase in activity. The enzyme was stimulated by SO4(2-), and kinetic experiments showed that SO4(2-) markedly increased the affinity of enzyme toward substrates: in the presence and absence of 0.35 M SO4(2-), the apparent Km values of hagfish phosphorylase b were 0.04 and 1.3% for glycogen, 8.7 and 66 mM for glucose 1-phosphate, and 0.05 and 1.0 mM for AMP, respectively. Electrophoretic and immunological data indicated that the hagfish possessed a single molecular form of phosphorylase, like the lamprey. Some immunological relatedness between the hagfish enzyme and the enzyme from lamprey or skate was demonstrated.

Animals↗
PubMedDate not supplied

Kinetic analysis and modelling of the allosteric behaviour of liver and muscle glycogen phosphorylases.

Allosteric enzymes have very complex kinetic behaviours which are primarily interpreted through simplified models. To describe the functional properties of liver and muscle glycogen phosphorylase isozymes we have developed an experimental strategy based on the measurements of initial reaction rates in the presence of different concentrations of the effectors glucose-1-phosphate and methyl-xanthines. Using the extensive structural information available for the two glycogen phosphorylase conformers T (inactive) and R (active) with different ligands, we have applied the Monod-Wyman-Changeux model and analysed the results in the context of the exclusive binding of the inhibitors to the T state, meanwhile the substrate glucose-1-phosphate binds to both, the R and T states. The kinetic analysis shows a good agreement between our model and the results obtained from the glycogen phosphorylases and inhibitors included in this study, which demonstrates the validity of the approach described here.

Allosteric Regulation↗
PubMed1994

Electrostatic effects in the control of glycogen phosphorylase by phosphorylation.

Electrostatic effects are important in the initial activation mechanism of glycogen phosphorylase by phosphorylation. Analysis of the electrostatic surface potential of glycogen phosphorylase with the program GRASP shows that in the unphosphorylated state, the N-terminal 20 residues, which include a number of basic amino acids, are located close to a position on the surface of the molecule that is highly acidic. Upon phosphorylation by phosphorylase kinase at Ser 14, the N-terminal residues change their position and conformation so that the Ser-P is directed away from the acidic patch and to an intersubunit site where 2 arginines bind the phosphate. This recognition site is created through tertiary and quaternary structural changes that accompany the activation mechanism.

Computer Simulation↗
PubMed1999

A recombinant form of the catalytic subunit of phosphorylase kinase that is soluble, monomeric, and includes key C-terminal residues.

Residues 302-326 of the catalytic (gamma) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKgamma was expressed using Escherichia coli. This yielded two stable, high-activity PhKgamma forms (35 and 42 kDa by SDS-PAGE) that were smaller than an authentic sample of rabbit muscle PhKgamma (45 kDa by SDS-PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKgamma residues 1-362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact gamma subunit. This implied that the lack of 25 PhKgamma C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKgamma that consisted of residues 1-300, denoted gamma1-300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.

Adenosine Triphosphate↗
PubMed1980

Spin-labelling of phosphorylase b using a paramagnetic 1-fluoro-2,4-dinitrobenzene derivative.

Phosphorylase b (1,4-alpha-D-glucan:1,6-alpha-D-glucan 6-alpha-glucosyltransferase, EC 2.4.1.1) can be specifically spin-labelled at a site essential for the catalytic action of the enzyme. A paramagnetic analogue of 1-fluoro-2,4-dinitrobenzene was synthesized and used as a dinitrophenylating agent. Reaction of phosphorylase b with the paramagnetic probe combined with the thiolysis method, leads to spin-labelling of a single -NH2 group (0.75 groups per subunit) with concomitant loss of 50% of the catalytic activity. Dinitrophenylation does not change the sedimentation profile of the enzyme. The ESR spectrum of modified phosphorylase b indicates that the attached label has rather limited segmental mobility and its environment is slightly hydrophobic. Small but subtle conformational changes induced by ligands in this critical site of the macromolecule can be directly detected by the spin-label. Also, sulfhydryl group modification of the spin-labelled enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) has a pronounced effect on the resonance spectrum.

Animals↗
PubMed1989

Role of calcium in the phenylephrine-induced activation of phosphorylase "A" in isolated liver cells.

The ability of phenylephrine to activate phosphorylase in liver cells with variable degrees of Ca2+ loading was studied. Phenylephrine has been found to be capable of stimulating phosphorylase at saturating Ca2+ concentrations that precluded any further action of this ion. Furthermore the degree of activation was proportional to the cellular calcium content. These results allow to conclude that alpha-adrenergic agonists activate phosphorylase by a mechanism apparently unrelated to their ability to mobilize and subsequently increase the cytosolic concentration of free Ca2+.

Animals↗
PubMed1988

Effects of quinacrine on vasopressin-induced changes in glycogen phosphorylase activity, Ca2+ transport and phosphoinositide metabolism in isolated hepatocytes.

In isolated hepatocytes, quinacrine (150-250 microM) inhibited vasopressin-induced increases in glucose release, glycogen phosphorylase a activity and 45Ca2+ efflux; and glucagon-induced increases in glucose release and cyclic AMP formation. These results indicate that a phospholipase A2 enzyme sensitive to quinacrine is unlikely to be involved in the process by which vasopressin stimulates glycogen phosphorylase activity in the liver cell. In cells labelled with [3H]inositol, much lower concentrations of quinacrine (20-50 microM) inhibited the stimulation by vasopressin of the accumulation of [3H]inositol. The drug had little effect on vasopressin-induced accumulation of [3H]inositol mono-, bis- and tris-phosphates. In the absence of vasopressin, higher concentrations of quinacrine caused a small stimulation of glycogen phosphorylase activity, 45Ca2+ release and the formation of [3H]inositol polyphosphates. Quinacrine did not inhibit the degradation by liver homogenates of inositol 1-phosphate, inositol 4,5-bisphosphate or inositol 1,4,5-trisphosphate. It is concluded that concentrations of quinacrine comparable with those which inhibit phospholipase A2 [G.J. Blackwell, W.G. Duncombe, R.J. Flower, M.F. Parsons and J.R. Vane, Br. J. Pharmac. 59, 353-366 (1977)] inhibit the stimulation by vasopressin of inositol utilization without significantly affecting coupling between hormone receptors and adenyl cyclase or phosphoinositide-specific phosphodiesterase, the action of the phosphodiesterase, and the degradation of inositol triphosphate.

Animals↗
PubMed1986

Hormonal regulation of phosphorylase phosphatase activity in rat liver.

The effect of glucagon and insulin on rat liver phosphorylase phosphatase activity in vivo was investigated. The activity of phosphatase was found to decrease following the administration of glucagon and increase with insulin in a reversible manner. No change was detected in the activity of heat-stable phosphatase inhibitors in the hormone-treated samples. Liver protein kinases (regulatory subunit of cAMP-dependent protein kinase and/or Ca2+-dependent phosphorylase kinase) are suggested to regulate the activity of hepatic phosphorylase phosphatase (type 1 and 2A).

Animals↗
PubMed1991

Amylin activates glycogen phosphorylase in the isolated soleus muscle of the rat.

The pancreatic beta-cell hormone amylin acts in isolated rat skeletal muscle to decrease insulin-stimulated incorporation of glucose into glycogen. It also increases blood levels of lactate and glucose in fasted rats in vivo. However, it remained uncertain whether amylin exerts direct effects to stimulate muscle glycogenolysis. We now report that amylin caused a dose-dependent increase in activity of muscle glycogen phosphorylase in isolated rat soleus muscle by stimulating phosphorylase a. Insulin inhibited amylin-stimulated activation of phosphorylase. Effects of amylin to stimulate muscle glycogenolysis are consistent with observed effects of amylin in vivo and could be a major mechanism whereby amylin modulates carbohydrate metabolism.

Amyloid↗
PubMed1995

Does phosphorylase kinase control glycogen biosynthesis in skeletal muscle?

Immunoblotting as well as enzyme assays demonstrate the presence of the self-glucosylating protein, glycogenin, in the protein-glycogen complex, in the sarcoplasmic reticulum and in phosphorylase kinase. In all three compartments glycogenin occurs in different, albeit, defined glucosylated forms, which upon deglucosylation are converted into a 42 kDa form. We suggest that phosphorylase kinase might have a dual function in glycogen biogenesis: firstly, control of glycogen degradation in the protein-glycogen complex via phosphorylation of glycogen phosphorylase b; secondly, regulation of glycogen biosynthesis on the sarcoplasmic reticular membranes via phosphorylation and thereby inhibition of glycogen synthase.

Animals↗
PubMed1987

Effect of ligands on Drosophila phosphorylase a as monitored by its enzymic inactivation.

The dephosphorylation of Drosophila phosphorylase a with the catalytic subunit of fruit-fly protein phosphatase-1 was inhibited by AMP, IMP, ADP, ATP, glucose-6-P, glucose-1-P and UDPG. Glucose, caffeine and glycogen did not influence the reaction. The inhibitory effect of AMP was reduced by glucose and caffeine. The above ligands acted through the modification of phosphorylase a conformation. This conclusion was drawn from the ligands' effect on the dephosphorylation of phosphohistone by Drosophila phosphatase-1 and on the tryptic digestion of fruit-fly phosphorylase a.

Animals↗
PubMed1979

Thermodynamics of nucleotides binding to phosphorylase b.

The effects of several chemical modifications in the AMP molecule on its interaction with phosphorylase b are examined by microcalorimetry, equilibrium dialysis, light scattering and ultracentrifuge experiments. In this work we report the results obtained for eight AMP analogues corresponding to different substituents in the puric base or in the ribose, or to different positions of the phosphate. The thermodynamic properties of the interaction between the phosphorylase b and the above mentioned nucleotides are also reported. The following conclusions were obtained: a) Except for IMP and 2'dIMP all the nucleotides studied clearly show two types of binding sites in the enzyme. b) The interaction of the nucleotide with its weaker affinity binding site is highly dependent upon chemical alterations in the puric base. c) Both the amino group in C(6) and the N(1) of the adenine in the AMP seem to play an important role in the conformational transitions induced by the nucleotide on the enzyme. d) The tetramerization of phosphorylase b in the presence of 10(-2) M AMP and in the conditions of the ultracentrifuge experiments is drastically affected by modifications in the ribose-phosphate part of the AMP molecule.

Animals↗