Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PHOSPHORYLASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,027 records · Page 57Linked to original sources
PubMed1989

Species differences in substrate specificity of pyrimidine nucleoside phosphorylase.

To compare the activity of pyrimidine nucleoside phosphorylase (PNP), an enzyme involved in the metabolism of 5-fluorouracil (5-FU), we used uridine (Urd), deoxyuridine (dUrd), and thymidine (dThd) as substrates and human, rat, and mouse neoplastic and normal tissues. As PNP activity was higher in the tumor tissues than in the normal ones in all species examined, the level of PNP activity is expected to be one critical factor linked to the effectiveness of 5-FU. In rats and mice, the ratio of the activities of Urd, dUrd, and dThd was about 10:7:1, whereas in humans, the ratio was 1:30:20. The main enzyme of PNP is Urd phosphorylase in rodents and dThd phosphorylase in humans. Therefore, when examining the metabolism of 5-FU and its analogues for potential clinical application, human tissues should be used.

Animals↗
PubMed1992

The molecular mechanism for the tetrameric association of glycogen phosphorylase promoted by protein phosphorylation.

The allosteric transition of glycogen phosphorylase promoted by protein phosphorylation is accompanied by the association of a pair of functional dimers to form a tetramer. The conformational changes within the dimer that lead to the creation of a protein recognition surface have been analyzed from a comparison of the crystal structures of T-state dimeric phosphorylase b and R-state tetrameric phosphorylase a. Regions of the structure that participate in the tetramer interface are situated within structural subdomains. These include the glycogen storage subdomain, the C-terminal subdomain and the tower helix. The subdomains undergo concerted conformational transitions on conversion from the T to the R state (overall r.m.s. shifts between 1 and 1.7 A) and, together with the quaternary conformational change within the functional dimer, create the tetramer interface. The glycogen storage subdomain and the C-terminal subdomain are distinct from those regions that contribute to the dimer interface, but shifts in the subdomains are correlated with the allosteric transitions that are mediated by the dimer interface. The structural properties of the tetramer interface are atypical of an oligomeric protein interface and are more similar to protein recognition surfaces observed in protease inhibitors and antibody-protein antigen complexes. There is a preponderance of polar and charged residues at the tetramer interface and a high number of H-bonds per surface area (one H-bond per 130 A2). In addition, the surface area made inaccessible at the interface is relatively small (1,142 A2 per subunit on dimer to tetramer association compared with 2,217 A2 per subunit on monomer-to-dimer association).

Allosteric Regulation↗
PubMed1987

Intron/exon structure of the human gene for the muscle isozyme of glycogen phosphorylase.

The intron/exon organization of the human gene for glycogen phosphorylase has been determined. The segments of the polypeptide chain that corresponds to the 19 exons of the gene are examined for relationships between the three-dimensional structure to the protein and gene structure. Only weak correlations are observed between domains of phosphorylase and exons. The nucleotide binding domains that are found in phosphorylase and other glycolytic enzymes are examined for relationships between exons of the genes and structures of the domains. When mapped to the three-dimensional structures, the intron/exon boundaries are shown to be widely distributed in this family of protein domains.

Amino Acid Sequence↗
PubMed1994

Liver glycogen synthase, phosphorylase, and the glycogen concentration in rats given a glucose load orally: a 24-hour study.

Fasted rats were given 4 g/kg glucose orally. Synthase R (active forms), total synthase, and phosphorylase alpha activities, and hepatic glycogen, glucose 6-phosphate (glucose-6P), uridine diphosphoglucose (UDP-glucose), glucose, and plasma glucose concentrations were determined over the subsequent 24 h. The resulting glycogen concentration changes could be divided into three distinct phases. A glycogen synthetic phase (between 0 and 4 h), a stability phase (between 4 and 12 h), and a degradation phase (between 12 and 24 h). Synthase R activity increased rapidly and reached a maximum at 20 min. With the onset of glycogen synthesis it gradually decreased below the control values, reaching a nadir by 4 h. During the glycogen stability phase it gradually increased again up to the control value. It then remained stable during the subsequent glycogen degradation phase. Phosphorylase a activity did not change throughout the entire 24-h period. Glucose-6-P concentration increased almost twofold at 20 min. It then decreased but was above the control values at the 24th h. The plasma and hepatic glucose concentrations increased as expected after the glucose load. They then decreased but remained above the control value at all subsequent time points. In summary, the synthase R, phosphorylase a activities, or changes in the known allosteric modifiers of these enzymes could not explain the changes in glycogen concentration. The reasons for these discrepancies remain to be determined.

Administration, Oral↗
PubMed2001

The cyclin-dependent kinase (CDK) inhibitor flavopiridol inhibits glycogen phosphorylase.

Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.

Adenosine Monophosphate↗
PubMed1995

Cloning and expression of human uridine phosphorylase.

Using a mouse cDNA probe we have identified a human uridine phosphorylase cDNA clone from the cDNA library of a human colorectal tumor cell line, HCT116. The recombinant human uridine phosphorylase expressed in COS-7 cells demonstrated specific enzyme activity with uridine as the substrate; this activity was inhibited by the competitive inhibitor 2,2'-anhydro-5-ethyluridine. Northern blot analysis with the cDNA as a probe demonstrated high levels of mRNA expression in several tumor cell lines but very low level in normal cell, WI-38. The expression of uridine phosphorylase mRNA in HCT-116 cells was further enhanced by treating the cells with vitamin D3 and the inflammatory cytokines: tumor necrosis factor alpha, interleukin 1 alpha and interferon gamma.

Amino Acid Sequence↗
PubMed1997

Autosomal recessive liver phosphorylase kinase deficiency caused by a novel splice-site mutation in the gene encoding the liver gamma subunit (PHKG2).

To facilitate mutation analysis of patients with an autosomal recessive form of liver phosphorylase kinase deficiency, the genomic structure of the gene encoding the testis/liver gamma subunit (PHKG2) was established. The gene consist of 10 exons. The translation start site is located in exon 2. Analysis of DNA from two female siblings, affected with liver phosphorylase kinase deficiency, by exon specific amplification followed by direct sequencing, revealed a single donor splice site mutation in the PHKG2 gene, IVS4 + 1(g --> a). The mutation leads to the skipping of exon 4, which results in a frameshift, starting at nucleotide 272, a premature stop codon after 32 additional amino acids, and subsequent loss of the catalytic site. It is concluded that deficiency of phosphorylase kinase in liver of the patients is caused by the IVS4 + 1(g --> a) mutation. In the patients described here, this genotype is associated with development of liver fibrosis.

Amino Acid Sequence↗
PubMed1994

Hexose metabolism in pancreatic islets: glycogen synthase and glycogen phosphorylase activities.

The activity of glycogen synthase and glycogen phosphorylase was measured in rat pancreatic islet homogenates. For this purpose, the sensitivity of current radioisotopic procedures for the assay of these enzymes in liver extracts was increased by about two orders of magnitude. Even so, the measurement of glycogen synthase and phosphorylase in islet homogenates was hampered by a potent amylase-like activity, resulting in the hydrolysis of preformed or newly formed 14C-labeled glycogen. Acarbose suppressed the latter phenomenon which was found attributable to both minute contamination of isolated islets by acinar cells and genuine alpha-amylase activity in purified islet beta-cells. As measured by the more sensitive method in the presence of acarbose, the a/(a+b) ratio for glycogen synthase activity in islet homogenates was increased in islets preincubated in the presence as distinct from absence of D-glucose and decreased after preincubation with forskolin. These changes represented a mirror image of those evoked by D-glucose and forskolin in the a/(a+b) ratio for glycogen phosphorylase activity. It is concluded that glycogen synthesis and breakdown are regulated in the endocrine pancreas in a manner qualitatively comparable to that prevailing in hepatocytes, the possible participation of an amylase-like activity to glycogen metabolism in intact islet beta-cells requiring further investigation.

Acarbose↗
PubMed1997

Proximal regions of the catalytic gamma and regulatory beta subunits on the interior lobe face of phosphorylase kinase are structurally coupled to each other and with enzyme activation.

Phosphorylase kinase from skeletal muscle is a hexadecameric enzyme with the subunit composition (alphabeta gammadelta)4 and a mass of 1.3 x 10(6) Da. The catalytic gamma subunit and the remaining regulatory subunits are packed as a tetrahedral structure composed of two elongated, opposing (alphabeta gammadelta)2 octameric lobes. We show by immunoelectron microscopy with subunit-specific monoclonal antibodies that a portion of the beta subunit occurs on the interior face of the lobes at a region of inter-lobal interactions, and that at a proximal position slightly more central and distal on the interior lobe face lies the base (residues 277 to 290) of the helical domain of the catalytic core of the gamma subunit. Activation of the kinase by a variety of means caused similar increases in the binding to the holoenzyme of the monoclonal antibodies against these two regions of the beta and gamma subunits. Moreover, monovalent fragments of the antibodies against both regions stimulated the activity of the non-activated holoenzyme. Thus, the epitopes of the beta and gamma subunits recognized by the monoclonal antibodies are structurally coupled to each other and with the activation of phosphorylase kinase. Activation of the holoenzyme apparently involves the repositioning of the base of the catalytic domain of the gamma subunit and a proximal region of the beta subunit within the identified area on the interior face of the lobes of the tetrahedral phosphorylase kinase molecule.

Amino Acid Sequence↗
PubMed1999

Heterologous expression of 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability.

The gene for the extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus was expressed at a high level in Escherichia coli thus providing a basis for detailed structural and functional studies of the enzyme. The recombinant enzyme was purified to homogeneity by means of a heat treatment (10 min at 100 degrees C) and by a single affinity chromatography step. The appropriate expression vector and host strain were selected and the culture conditions were determined that would ensure a consistent yield of 6 mg of pure enzyme per liter of culture. The heterologously expressed enzyme is identical to the original S. solfataricus 5'-methylthioadenosine phosphorylase regarding molecular weight, substrate specificity, and the presence of intersubunit disulfide bonds. On the other hand, the recombinant 5'-methylthioadenosine phosphorylase is less thermophilic and thermostable than the S. solfataricus enzyme, since an incorrect positioning of disulfide bonds within the molecule generates structures less stable to thermal unfolding.

Base Sequence↗
PubMed1993

A quantitative histochemical procedure for the demonstration of purine nucleoside phosphorylase activity in rat and human liver using Tetranitro BT and xanthine oxidase as auxiliary enzyme.

A quantitative histochemical procedure was developed for the demonstration of purine nucleoside phosphorylase in rat liver using unfixed cryostat sections and the auxiliary enzyme xanthine oxidase. The optimum incubation medium contained 18% (w/v) poly(vinyl alcohol), 100 mM phosphate buffer, pH 8.0, 0.5 mM inosine, 0.47 mM methoxyphenazine methosulphate and 1 mM Tetranitro BT. An enzyme film consisting of xanthine oxidase was brought onto the object slides before the section wa allowed to adhere. The specificity of the reaction was proven by the low amount of final reaction product generated when incubating in the absence of inosine. Moreover, 1 mM p-chloromercuribenzoic acid, a non-specific inhibitor of purine nucleoside phosphorylase, inhibited the specific reaction by 90%. The specific reaction defined as the test reaction, in the presence of substrate, minus the control reaction, in the absence of substrate was linear with incubation time at least up to 30 min as measured cytophotometrically. A high activity was observed in endothelial cells and Kupffer cells of rat liver and a lower activity in liver parenchymal cells. Pericentral hepatocytes showed an activity higher than that of periportal hepatocytes. In human liver, purine nucleoside phosphorylase activity was also high in endothelial cells and Kupffer cells, but the activity in liver parenchymal cells was only slightly lower than it was in non-parenchymal cells. The localization of the enzyme is in agreement with earlier ultrastructural findings using fixed liver tissue and the lead salt procedure.

Animals↗
PubMed1992

Post-receptor defect accounts for phosphorylase hypersensitivity in cultured diabetic cardiomyocytes.

The basis for the hypersensitive response of glycogen phosphorylase to epinephrine stimulation was investigated in adult rat cardiomyocytes isolated from normal and alloxan-diabetic animals. To assess potential G-protein involvement in the response, normal and diabetic derived myocytes were incubated with either cholera or pertussis toxin prior to hormonal stimulation. Pretreatment of cardiomyocytes with cholera toxin resulted in a potentiated response to epinephrine stimulation whereas pertussis toxin did not affect the activation of this signaling pathway. To determine if the enhanced response of phosphorylase activation resulted from an alteration in adenylate cyclase activation, the cells were challenged with forskolin. After 3 hr in primary culture, diabetic cardiomyocytes exhibited a hypersensitive response to forskolin stimulation relative to normal cells. However, after 24 hr in culture, both normal and diabetic myocytes responded identically to forskolin challenge. The present data suggest that a cholera toxin sensitive G-protein mediates the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in diabetic cardiomyocytes and this response which is present in alloxan-diabetic cells and is induced in vitro in normal cardiomyocytes is primarily due to a defect at a post-receptor site.

Adenylate Cyclase Toxin↗
PubMed1990

Immunohistochemical demonstration of glycogen phosphorylase in rat brain slices.

Paraffin-embedded sections from paraformaldehyde-fixed rat brain were stained immunocytochemically for glycogen phosphorylase brain isozyme BB, using a monoclonal mouse antibody and the biotin-strept-avidin method, with either horseradish peroxidase or beta-galactosidase as marker enzymes. Two cell types showed strong glycogen phosphorylase-immunoreactivity: Astrocytes and ependymal cells. Most intensive staining was observed in the cerebellar cortex, the neocortex and the hippocampus. Astrocytes in the cerebellar white matter stained positively. The choroid plexus cells stained poorly or not at all. Neurons throughout the brain were negative, as well as oligodendrocytes and bundles of myelinated nerve fibers. These data are consistent with the immunocytochemical localization of glycogen phosphorylase in astroglia-rich primary cultures derived from rat brain.

Animals↗
PubMed1993

Glycogen phosphorylase activity and immunoreactivity during pre- and postnatal development of rat brain.

Catalytic activity and immunoreactivity of glycogen phosphorylase were studied in pre- and postnatal rat brain. The catalytic activity was assayed in brain homogenates; immunoreactivity was investigated by immunoblot analysis using a monoclonal anti-bovine brain glycogen phosphorylase antibody. The cellular localization and intensity of immunoreactivity were analysed on paraffin-embedded sections utilizing the same monoclonal antibody. The catalytic activity increased 10-fold from embryonic day 16 to adult; immunoreactivity became detectable on embryonic day 16 and increased in intensity as the enzyme activity rose to adult values. The first cellular elements to be stained immunohistochemically were ependymal cells lining the ventricles, ependymal cells of the choroid plexus, meningeal cells and a selected population of neurons in the brain stem. The immunoreactivity of plexus cells and meningeal cells was reduced or absent in the adult rat brain. The earliest appearance of glycogen phosphorylase immunoreactivity in astroglial cells was seen at postnatal day 9 in the hippocampus. The staining pattern of the adult brain was reached at day 22 post partum. The developmental changes in glycogen deposition and in glycogen phophorylase activity and immunoreactivity may indicate a variable physiological role of glycogen metabolism for different cell types in the pre- and postnatal periods.

Animals↗
PubMed1980

Altered purine and pyrimidine metabolism in erythrocytes with purine nucleoside phosphorylase deficiency.

Purine and pyrimidine metabolism was compared in erythrocytes from three patients from two families with purine nucleoside phosphorylase deficiency and T-cell immunodeficiency, one heterozygote subject for this enzyme deficiency, one patient with a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase, and two normal subjects. The erythrocytes from the heterozygote subject were indistinguishable from the normal erythrocytes. The purine nucleoside phosphorylase deficient erythrocytes had a block in the conversion of inosine to hypoxanthine. The erythrocytes with 0.07% of normal purine nucleoside phosphorylase activity resembled erythrocytes with hypoxanthine-guanine phosphoribosyltransferase deficiency by having an elevated intracellular concentration of PP-ribose-P, increased synthesis of PP-ribose-P, and an elevated rate of carbon dioxide release from orotic acid during its conversion to UMP. Two hypotheses to account for the associated immunodeficiency--that the enzyme deficiency leads to a block of PP-ribose-P synthesis or inhibition of pyrimidine synthesis--could not be supported by observations in erythrocytes from both enzyme-deficient families.

Child↗
PubMed1975

Enzyme activity during the metabolism of glycogen. I. Demonstration of phosphorylases in the sensory cells of the tuberous organ of Gnathonemus petersii (Mormyridae). Histochemical and ultrastructural methods.

Phosphorylase activities were investigated by histochemical and ultrastructural procedures in the electroreceptive sensory cells of the tuberous organ of Gnathonemus petersii. Ater incubation in G1P, G1P activated by AMP (Takeuchi and Kuriaki medium) or in G1P activated by ATP+MgSO4 (Guha and Wegman medium) newly formed polysaccharides were analysed with the iodine and P.A.S. reactions under light microscopy and, under electron microscopy, with the periodic acid thiocarbohydrazide (TCH) silver proteinate (PATAg reaction, Thiery), The newly formed polysaccharides proved the presence of glycogen phosphorylase (2.4.1.1) activities and of their branching enzymes (2.4.1.18). When G1P was activated by ATP+MgSO4, they appeared as glycogen particles with the same constitution as native glycogen. After incubation in G1P and in G1P activated by AMP they appeared as glycogen and polyglucose filaments too. In the latter case they were high concentrated. The results show that the phosphorylases are principally present in this sensory cell in their inactive form.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1980

In vivo synthesis of glycogen by phosphorylase system.

Application of conventional histochemical techniques to the living chick retina demonstrates that phosphorylase can synthesize glycogen (polyglucose) in vivo, in the paraboloid of the accessory cone. Natural in vivo glycogen synthesis may therefore be due to glycogen synthetase and phosphorylase systems, although phosphorylase is normally regarded as a glycolytic enzyme.

Animals↗
PubMed1983

Genetic variability of purine nucleoside phosphorylase activity in the mouse: relationship to Np-1 and Np-2.

A survey of 37 inbred strains for erythrocyte purine nucleoside phosphorylase activity showed a greater than threefold range. Six of these strains had significantly greater activity than the others, and all of the high-activity strains had the Np-2 electrophoretic band. The high-purine nucleoside phosphorylase activity trait corresponding to Np-2 was inherited in an autosomal codominant manner and minor differences were apparent in thermal and kinetic properties between low- and high-activity strains. This work provides further support for there being either two structural loci for purine nucleoside phosphorylase, Np-1 and Np-2, or a regulatory-modifier locus.

Animals↗