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At least 1,261 records · Page 70Linked to original sources
PubMed1996

Interaction of muscle glycogen phosphorylase b reconstituted from apoenzyme and analogs of pyridoxal-5'-phosphate with specific ligands.

Phosphorylase b from rabbit skeletal muscles was reconstituted with analogs of PLP containing residues -CH(2)-CH(2)-COOH, trans-CH=CH-COOH or -C=-COOH at position 5. Replacing native coenzyme in the phosphorylase molecule with any PLP analog tested leads to the decrease in the enzyme affinity for the allosteric inhibitor, FMN. Phosphorylase b reconstituted with analogs of PLP shows the greater ability for association in tetramers in the presence of 1 mM AMP than native enzyme.

Animals↗
PubMed1997

Use of hydrated reversed micelles of surfactant in organic solvent for stabilization of individual oligomeric forms of uridine phosphorylase from Escherichia coli K-12.

The catalytic activity of uridine phosphorylase from Escherichia coli K-12 entrapped in hydrated reversed micelles of aerosol OT (AOT) in octane has been studied as a function of the degree of hydration of the micelles. It was shown that the catalytic activity of uridine phosphorylase reached maximum values at [H2O/[AOT] ratios equal to 8.4, 12.9, 16.1 and 18.6. Based on the sedimentation data the conclusion has been made that the maxima of the catalytic activity correspond to the monomeric, dimeric, trimeric and tetrameric forms of the enzyme. The measurements of the rate of the enzymatic reaction catalyzed by uridine phosphorylase entrapped in hydrated reversed micelles at various concentrations of AOT indicate that the monomeric enzyme form, in contrast to the trimeric and tetrameric forms, exhibits the membranotropic properties.

Aerosols↗
PubMed1998

Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors.

Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. We purified the enzyme from the murine colon 26 tumor using a two-step procedure through 5-amino-benzylacyclouridine affinity chromatography. Antibodies raised in rabbits against the purified protein revealed single bands in Western blots of normal human tissue and tumor extracts. The polyclonal antibody used to screen a human liver expression library allowed the isolation of a 1.2-kb clone that contained the entire open reading frame of the human UPase. The UPase cDNA has been expressed as a fusion protein in Escherichia coli using the pMal-C2 vector. The kinetic analysis demonstrated that the recombinant UPase preferentially uses uridine, 5-fluorouracil, and uracil as substrates, although lower levels of activity were observed with 2-deoxyuridine and thymidine. Clinical samples of human tumors and adjacent normal tissues were assayed for phosphorolytic activity and sensitivity to 5-benzylacyclouridine (BAU), a potent inhibitor of the enzyme presently in Phase I-II clinical trial. Activity in normal tissues appeared to be low but very sensitive to BAU (approximately 90% inhibition at 10 microM). Tumors had generally 2-3-fold greater activity compared with adjacent normal tissues. In breast cancer specimens and head-neck squamous carcinomas, however, uridine cleavage was only partially inhibited (40-60%) by 10 or 100 microM BAU. The BAU-insensitive activity requires phosphate and pH conditions similar to the normal enzyme, and the new phosphorolytic activity was independent from thymidine phosphorylase. The BAU-insensitive phosphorolytic activity in selected tumors, coupled with the potent inhibitory activity of BAU against the "classical" uridine phosphorylase in normal human tissues, provides the rationale for combining BAU with 5-fluorouracil in the treatment of breast and head-neck tumors.

Amino Acid Sequence↗
PubMed1986

Thermodynamics of the binding of AMP to glycogen phosphorylase a.

The binding of AMP to rabbit muscle glycogen phosphorylase a (EC 2.4.1.1.) has been studied by equilibrium dialysis and isothermal microcalorimetry at pH 6.9 over a temperature range of 25 degrees C to 35 degrees C. Thermal titration experiments were carried out in various buffer systems. We have found by these methods that a certain number of protons are released when the protein binds to the ligand and are taken up by the buffer. The tetramer of phosphorylase a has been shown to have four equal and independent, non-cooperative binding sites for AMP at 25 degrees C, 30 degrees C, and 35 degrees C; these sites can be assigned to the so-called nucleotide or, activator, sites in the protein. The binding constants together with the changes in Gibbs energy, enthalpy, and entropy per site for the AMP binding were calculated at each temperature. A negative delta Cp value of -2.3 +/- 0.2 J K-1 (AMP bound)-1 was obtained for this binding process. The hydrophobic and vibrational contributions of the heat capacity and entropy changes have been resolved by the method described by Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). From this analysis, it appears that the binding is, in all cases, enthalpy-driven, the two entropic contributions, hydrophobic and vibrational, having opposing effects.

Adenosine Monophosphate↗
PubMed1988

Activity of glycogen synthase and phosphorylase and glucose 6-phosphate content in renal clear cell carcinomas.

Eleven renal clear cell carcinomas were investigated with regard to their glycogen and glucose 6-phosphate (G6P) content and the activities of glycogen synthase and phosphorylase. Compared to normal cortex from the same kidneys, all tumours revealed increased glycogen and G6P content. The activities of phosphorylase a and b as well as of synthase R and D were also increased. Synthase I activity, however, was reduced. It was concluded that in these tumours glycogen synthesis may preferentially be catalyzed by synthase R which is activated by G6P. These findings agree with those in some other glycogenotic tissues and support the concept that an accumulation of G6P is associated with excessive storage of glycogen in preneoplastic and neoplastic lesions.

Adenocarcinoma↗
PubMed1991

Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP.

The crystal structures of activated R state glycogen phosphorylase a (GPa) and R and T state glycogen phosphorylase b (GPb) complexed with AMP have been solved at 2.9 A, 2.9 A and 2.2 A resolution, respectively. The structure of R state GPa is nearly identical to the structure of sulphate-activated R state GPb, except in the region of Ser14, where there is a covalently attached phosphate group in GPa and a non-covalently attached sulphate group in GPb. The contacts made by the N-terminal tail residues in R state GPa at the subunit interface of the functionally active dimer are similar to those observed previously for T state GPa. The quaternary and tertiary structural changes on the T to R transition allow these interactions to be relayed to the catalytic site in R state GPa. The transition from the T state GPb structure to the R state GPa structure results in a change in the N-terminal residues from a poorly ordered extended structure that makes intrasubunit contacts to an ordered coiled conformation that makes intersubunit contacts. The distance between Arg10, the first residue to be located from the N terminus, in R state GPa and T state GPb is 50 A. One of the important subunit-subunit interactions in the dimer molecule involves contacts between the helix alpha 2 and the cap' (residues 35' to 45' that form a loop between the 1st and 2nd alpha helices, alpha 1' and alpha 2' of the other subunit. The prime denotes residues from the other subunit). The interactions made by the N-terminal residues induce structural changes at the cap'/alpha 2 helix interface that lead to the creation of a high-affinity AMP site. The tertiary structural changes at the cap (shifts 1.2 to 2.1 A for residues 35 to 45) are partially compensated by the quaternary structural change so that the overall shifts in these residues after the combined tertiary and quaternary changes are between 0.5 and 1.3 A. AMP binds to R state GPb with at least 100-fold greater affinity and exhibits four additional hydrogen bonds, stronger ionic interactions and more extensive van der Waals' interactions with 116 A2 greater solvent accessible surface area buried compared with AMP bound to T state GPb.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Monophosphate↗
PubMed1983

Purification and characterization of phosphorylase B from Ascaris suum.

Glycogen phosphorylase b (EC 2.4.1.1) has been purified from the muscle of the roundworm, Ascaris suum. The 223-fold purified enzyme was shown to be homogenous by high performance liquid chromatography (HPLC), gel filtration column chromatography and sodium dodecyl sulfate (SDS) gel electrophoresis. The apparent native molecular weight of the enzyme determined by size exclusion chromatography by HPLC and gel filtration corresponded to 200 000 and 199 000, respectively. The subunit molecular weight of the enzyme was determined to be 100 000 by electrophoresis in the presence of SDS. Therefore, the enzyme appears to be a dimer with identical or near identical subunits. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol subunit and exhibited an absorbance index E1% 280 of 13.8. The apparent isoelectric point of the enzyme is 5.53. The enzyme, inactive in the absence of AMP, can be converted to the active form by rabbit muscle phosphorylase kinase and MgATP. The molecular weight of the activated form of the enzyme is 200 000. Kinetic studies showed apparent Km values of 0.17% for glycogen, 36 mM for Pi and 52 mM for glucose-1-P. The apparent Ka for AMP was 0.22 mM.

Animals↗
PubMed2005

Modeling aided design of potent glycogen phosphorylase inhibitors.

Molecular modeling has been used to assist in the development of a novel series of potent glycogen phosphorylase inhibitors based on a phenyl diacid lead, compound 1. In the absence of suitable competitive binding assays, compound 1 was predicted to bind at the AMP allosteric site based on superposition onto known inhibitors which bind at different sites in the enzyme and analyses of the surrounding protein environment associated with these distinct sites. Possible docking modes of compound 1 at the AMP allosteric site were further explored using the crystal structure of rabbit muscle glycogen phosphorylase complexed with a Bayer diacid compound W1807 (PDB entry 3AMV). Compound 1 was predicted to interact with positively charged arginines at the AMP allosteric site in the docking model. Characterization of the binding pocket by a grid-based surface calculation of the docking model revealed a large unfilled hydrophobic region near the central phenyl ring, suggesting that compounds with larger hydrophobic groups in this region would improve binding. A series of naphthyl diacid compounds were designed and synthesized to access this hydrophobic cleft, and showed significantly improved potency.

Adenosine Monophosphate↗
PubMed1990

[Glycogen phosphorylase from human leukocytes. Isolation and kinetic properties].

Homogeneous glycogen phosphorylase from human leukocytes has been obtained. A one-step bioluminescent procedure for the enzyme activity assay has been developed. This method is based on a continuous recording of the product of the glycogen phosphorylase-catalyzed reaction using a coimmobilized multienzyme system (phosphoglucomutase, glucose-6-phosphate dehydrogenase, NADH:FMN oxidoreductase and bacterial luciferase). The method sensitivity is 10 times as high compared to earlier described methods. The Km values for glycogen (0.2 mg/ml) and phosphate (3.9 mM) at pH 7.9 were determined. AMP was shown to be the enzyme effector.

Animals↗
PubMed1979

[Regulation of glycogen metabolism in the liver and hepatic glycogenosis due to phosphorylase system deficiency].

Glycogen synthesis and breakdown in the liver are tightly controlled through different mechanisms. The purpose of this review is to describe some properties of the enzymes involved in the glycogen metabolism and the sequence of events by which glucose, allosteric effectors and hormones control this metabolism in the liver. Clinical, genetic and biological aspects of the phosphorylase and the phosphorylase kinase deficiencies are examined. The enzymatic analysis of the haemolysates from the patients allows discrimination of these two types of glycogenosis.

Glycogen Storage Disease↗
PubMedDate not supplied

[Glycogen content and activities of phosphorylase and glucose-6-phosphatase in the fast and slow muscles of representatives of different classes of vertebrates].

Studies have been made on glycogen content as well as on the activity of phosphorylase and glucose-6-phosphatase in fast and slow muscles from representatives of 6 classes of vertebrates (Lampetra fluviatilis, Cyprinus carpio, Rana temporaria, Rana ridibunda, Emys orbicularis, hen, rat). Glycogen level and glucose-6-phosphatase activity are either higher in slow muscles, or practically identical in both types of muscles (glucose-6-phosphatase is absent from the fast muscles of hens and rats). On the contrary, phosphorylase activity is higher in fast muscles, this finding being true only for higher vertebrates and lampreys.

Animals↗
PubMed1992

E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism.

It has been previously shown that the pnp messenger RNAs are cleaved by RNase III at the 5' end and that these cleavages induce a rapid decay of these messengers. A translational fusion between pnp and lacZ was introduced into the chromosome of a delta lac strain to study the expression of pnp. In the presence of increased cellular concentrations of polynucleotide phosphorylase, the level of the hybrid beta-galactosidase is repressed, whereas the synthesis rate of the corresponding message is not significantly affected. In the absence of pnp, the level of the hybrid protein increases strongly. Thus, polynucleotide phosphorylase is post-transcriptionally autocontrolled. However, autocontrol is totally abolished in strains where the RNase III site on the pnp message has been deleted or in strains devoid of RNase III. These results suggest that polynucleotide phosphorylase requires RNase III cleavages to autoregulate the translation of its message. Other mutations in the ribosome binding site region support the hypothesis that this 3' to 5' processive enzyme could recognize a specific repressor binding site at the 5' end of pnp mRNA. Implications of these results on the mechanism of regulation and on messenger degradation are discussed.

Base Sequence↗
PubMed1993

Purification and properties of laminaribiose phosphorylase (EC 2.4 1.31) from Euglena gracilis Z.

Three isoforms of laminaribiose phosphorylase, F0, F1, and F2, were purified to an electrophoretically homogeneous state from a cell free extract of Euglena gracilis Z (IAM E-6). F1 and F2 were the major components. The three enzymes showed very similar properties except their isoelectric points. They could also use laminaribiose as the glucosyl acceptor instead of glucose. The isoforms the same molecular mass of 120 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) or 200 kDa by the gel filtration method, suggesting that they have a dimer structure. They could not be distinguished on the Ouchterlony's double diffusion test with mouse antiserum against F1 or F2. The substrate specificities of F1 and F2 were determined to be essentially the same. Both of the reaction mechanisms of F1 and F2 were determined to be ordered bi-bi mechanisms, as in the case of cellobiose phosphorylase. A competitive substrate inhibition was observed in their synthetic reaction. Two other strains, E. gracilis var. bacillaris (IAM E-2) and E. gracilis (IAM E-3), had only one laminaribiose phosphorylase, which corresponded to F0 on native PAGE.

Animals↗
PubMed2000

Impaired glycogen synthesis in hepatocytes from Zucker fatty fa/fa rats: the role of increased phosphorylase activity.

AIMS/HYPOTHESIS: The Zucker fatty fa/fa rat develops hyperinsulinaemia, insulin-resistance and severe obesity as a result of a homozygous mutation in the leptin receptor gene. The aim was to characterise the metabolic defect(s) in hepatocytes from fa/fa rats. METHODS: Glucose metabolism and key regulatory enzymes were investigated in hepatocytes from fa/fa and Fa/? rats after short-term culture in the absence of insulin. RESULTS: Hepatocytes from fa/fa rats have higher glucokinase activity and expression of the glucokinase regulatory protein and higher rates of glycolysis and lipogenesis, but lower rates of glycogen synthesis than hepatocytes from Fa/? controls. Insulin caused a similar stimulation of glycogen synthesis in hepatocytes from fa/fa rats as in controls ( > twofold) but did not restore the impaired glycogen synthesis in cells from fa/fa rats. Adenovirus-mediated glucokinase overexpression stimulated glycogen synthesis and glycolysis but aggravated rather than abolished the relative impairment of glycogen synthesis in cells from fa/fa rats. Inhibition of glycolysis with 2,5-anhydromannitol, an inhibitor of glycolysis and gluconeogenesis, increased glucose 6-phosphate concentrations and glycogen synthesis in hepatocytes from Fa/? and fa/fa rats but did not restore the impaired glycogen synthesis in cells from fa/fa rats. Hepatocytes from fa/fa rats had a higher activity of phosphorylase a in the basal state and after incubation with insulin or glucagon and higher total phosphorylase. CONCLUSION/INTERPRETATION: The increased activity of phosphorylase is a major contributing factor to the impaired glycogen synthesis in hepatocytes from fa/fa rats and could contribute to the lipogenic state by a glycogenolytic-glycolytic-lipogenic pathway.

Animals↗
PubMed1984

A one-step enzymatic assay for sucrose with sucrose phosphorylase.

A one-step, enzymatic assay for sucrose using sucrose phosphorylase is described. Sucrose phosphorylase, which is now commercially available, was isolated from Leuconostoc mesenteroides strain B-1200 and partially purified by ammonium sulfate precipitation. Samples containing 5 to 80 nmol of sucrose are mixed with potassium phosphate, NAD, sucrose phosphorylase, and two commercial enzymes, phosphoglucomutase and NAD-accepting glucose-6-phosphate dehydrogenase. After 30 min incubation at room temperature, absorbance at 340 nm is proportional to initial sucrose content. A 20-fold molar excess of glucose or a twofold excess of fructose have no effect on the assay, while a fourfold excess of fructose interferes with the assay by decreasing absorbance ca. 20%. This assay was designed to provide a rapid method for determining sucrose in studies of sugar transport by plants. To test the assay, corn pedicel extracts were assayed enzymatically and by high-pressure liquid chromatography. Estimates of sucrose content made by the two methods were equivalent, and exogenous addition of sucrose to these samples resulted in the expected increase in apparent sucrose content.

Chromatography, High Pressure Liquid↗
PubMed1990

Substrate-induced activation of maltose phosphorylase: interaction with the anomeric hydroxyl group of alpha-maltose and alpha-D-glucose controls the enzyme's glucosyltransferase activity.

Maltose phosphorylase, long considered strictly specific for beta-D-glucopyranosyl phosphate (beta-D-glucose 1-P), was found to catalyze the reaction beta-D-glucosyl fluoride + alpha-D-glucose----alpha-maltose + HF, at a rapid rate, V = 11.2 +/- 1.2 mumol/(min.mg), and K = 13.1 +/- 4.4 mM with alpha-D-glucose saturating, at 0 degrees C. This reaction is analogous to the synthesis of maltose from beta-D-glucose 1-P + D-glucose (the reverse of maltose phosphorolysis). In acting upon beta-D-glucosyl fluoride, maltose phosphorylase was found to use alpha-D-glucose as a cosubstrate but not beta-D-glucose or other close analogs (e.g., alpha-D-glucosyl fluoride) lacking an axial 1-OH group. Similarly, the enzyme was shown to use alpha-maltose as a substrate but not beta-maltose or close analogs (e.g., alpha-maltosyl fluoride) lacking an axial 1-OH group. These results indicate that interaction of the axial 1-OH group of the disaccharide donor or sugar acceptor with a particular protein group near the reaction center is required for effective catalysis. This interaction appears to be the means that leads maltose phosphorylase to promote a narrowly defined set of glucosyl transfer reactions with little hydrolysis, in contrast to other glycosylases that catalyze both hydrolytic and nonhydrolytic reactions.

Binding Sites↗
PubMed1991

The phosphate recognition site of Escherichia coli maltodextrin phosphorylase.

The role of two positively charged amino acid residues located at the active site of Escherichia coli maltodextrin phosphorylase was investigated by site-directed mutagenesis. Substitution of Lys539 by an arginine caused a 600-fold reduction, substitution of Arg534 by a glutamine caused an even larger 7000-fold reduction of the catalytic rate while substrate binding remained essentially unaffected. Since the Arg534----Gln exchange reduces the catalytic rate near to inactivity and even the conservative Lys534----Arg exchange caused a marked decrease of activity, the central functional role of both positively charged residues in phosphorylase catalysis anticipated by the crystallographic analysis of the corresponding amino acid residues Arg569 and Lys574 in the catalytic site of phosphorylase b was confirmed.

Amino Acid Sequence↗
PubMed1995

Production of trehalose phosphorylase by Catellatospora ferruginea.

A range of microorganisms was screened for new and high producer strains of trehalose phosphorylase (EC 2.4.1.64). Trehalose phosphorylase activity was found in cells of actinomyctes of the genera Actinomadura, Amycolata, Catellatospora, Kineosporia, and Nocardia. Among them, Catellatospora ferruginea showed the highest enzyme activity. Trehalose phosphorylase from C. ferruginea was able to catalyse both the phosphorolysis of trehalose into beta-glucose 1-phosphate and D-glucose and the synthesis of trehalose from beta-glucose 1-phosphate and D-glucose.

Actinomycetaceae↗