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PubMed1991

Interaction of salmon glucagon, glucagon-like peptide, and epinephrine in the stimulation of phosphorylase a activity in fish isolated hepatocytes.

The simultaneous addition of epinephrine and salmon glucagon to catfish (Ictalurus melas) and trout (Salmo gairdneri) hepatocytes did not induce greater increases in glycogen phosphorylase a activity and in glucose release than those caused by epinephrine alone. The effects of epinephrine are greater than those of glucagon. Propranolol added to the hormonal pool blocked the epinephrine effects. In trout cells, epinephrine and glucagon-like peptide (GLP) had similar effects and when they were added simultaneously the stimulation of metabolic indices was higher compared to that obtained with either epinephrine or GLP. However, the effects were not additive. In the presence of epinephrine plus GLP the inhibitory effect of propranolol was not evident, due to the effect induced by GLP, on which propranolol was not effective. This may indicate that epinephrine masks the GLP effect. Results could mean that epinephrine and glucagon-family peptides act in catfish and trout hepatocytes through different receptors on the same pathway leading to glycogen phosphorylase a activation.

Animals↗
PubMed1990

Purification and characterization of native and proteolytic forms of rabbit liver phosphorylase kinase.

1. Two forms of phosphorylase kinase having mol. wt of 1,260,000 (form I) and 205,000 (form II) have been identified by gel filtration chromatography of rabbit liver crude extracts. 2. Form I was the majority when the homogenization buffer was supplemented with a mixture of proteinase inhibitors. This form has been purified through a protocol including ultracentrifugation, gel filtration and affinity chromatography on Sepharose-heparin. 3. Form II was purified by a combination of chromatographic procedures including ion exchange, gel filtration and affinity chromatography on Sepharose-Blue Dextran and Sepharose-histone. 4. Upon electrophoresis in the presence of sodium dodecyl sulfate two subunits of 69,000 and 44,000 were identified for this low molecular weight enzyme. Thus, a tetrameric structure comprising two subunits of each kind can be proposed. 5. Treatment of form I with either trypsin or chymotrypsin gave an active fragment having a molecular weight similar to that of form II. On the contrary, other dissociating treatments with salts, thiols and detergents failed in producing forms of lower molecular weight. 6. The similarities between proteolyzed forms I and II were stressed by their behavior in front of antibodies raised against the muscle isoenzyme of phosphorylase kinase. 7. The study of the effect of magnesium and fluoride ions on the activity of both forms showed an inhibitory effect of magnesium when its concentration exceeded that of ATP. 8. The inhibition could nevertheless be reverted by including 50 mM NaF in the reaction mixture. 9. Form I and form II could be distinguished by their pH dependence in the presence of an excess of magnesium ions over ATP, whereas the affinity for both substrates was not significantly different.

Adenosine Triphosphate↗
PubMed1980

Immunofluorescence: a sensitive and rapid method for the detection of purine nucleoside phosphorylase in single cells.

Indirect immunofluorescence was used as a rapid, sensitive and specific method for the visualization of the enzyme purine nucleoside phosphorylase in single cells. The enzyme was localized throughout cytoplasm of human lymphoblasts and fibroblasts but not in cell nuclei. This method is valuable for the detection of mutant enzyme protein in cell-mediated immunodeficiencies caused by purine nucleoside phosphorylase deficiency since it does not rely on enzyme activity. It requires only a limited number of cells and can therefore be used for the rapid screening for the presence of cross-reactive protein in immunodeficiency diseases.

Female↗
PubMed1987

Hepatic glycogen synthase phosphatase and phosphorylase phosphatase activities are increased in obese (fa/fa) hyperinsulinemic Zucker rats: effects of glyburide administration.

The chronically hyperinsulinemic Zucker fatty rat, with peripheral insulin resistance and glucose intolerance, represents a model of noninsulin dependent diabetes mellitus (NIDDM). These animals have elevated hepatic glycogen levels. Hepatic levels of synthase phosphatase and phosphorylase phosphatase, which are diminished in the IDDM rat, were markedly increased in the obese rats. Glyburide, a sulfonylurea used in treatment of NIDDM, resulted in reduced levels of glycemia and increased insulin levels in Zucker rats. Hepatic glycogen levels were increased, as was the activation of glycogen synthase, although there were no effects of drug administration on synthase phosphatase or phosphorylase phosphatase activities. G6P levels were increased by glyburide in lean rats but not in obese animals. These effects of glyburide on liver glycogen metabolism are accounted for via potentiation of the glycogenic effects of insulin.

Animals↗
PubMed1989

Effect of sulfonylureas on hepatic glycogen metabolism: activation of glycogen phosphorylase.

In hepatocytes isolated from fed rats, both tolbutamide and glipizide caused a dose-dependent activation of glycogen phosphorylase, possibly by a Ca2+-mediated mechanism. Maximal effects (about twofold) were already obtained when drugs were used at 0.5 mmol/L, the calculated concentrations of tolbutamide and glipizide responsible for the half-maximal effects being 60 and 30 mumol/L, respectively. The activation of glycogen phosphorylase caused the mobilization of glycogen and increased the cellular concentration of hexose 6-phosphates (glucose 6-phosphate plus fructose 6-phosphate) and that of fructose 2,6-bisphosphate. Under the influence of sulfonylureas, glucose formation was slightly stimulated while the rate of L-lactate production was more markedly incremented, indicating that sulfonylureas canalize the metabolic flux coming from glycogen mainly to the glycolytic pathway. These results suggest that a glycogenolytic action of sulfonylureas could collaborate to raise hepatic fructose 2,6-bisphosphate concentration in the fed animal.

Animals↗
PubMed1995

Modulation of glycogen phosphorylase activity and fructose 2,6-bisphosphate levels by glibenclamide and meglitinide in isolated rat hepatocytes: a comparative study.

The influence of glibenclamide and meglitinide, or 4-[2-(5-chloro-2-methoxybenzamide)ethyl]-benzoic acid, a compound similar to the nonsulfonylurea moiety of glibenclamide, on glycogen phosphorylase a activity, fructose 2,6-bisphosphate (F-2,6-P2) level, and cytoplasmic free-Ca2+ concentration has been studied in isolated rat hepatocytes. Both glibenclamide and meglitinide caused a transient and dose-dependent activation of glycogen phosphorylase, with half-maximal effects corresponding to 3.7 +/- 1.6 and 9.6 +/- 3.3 mumol/L, respectively. This enzyme activation occurred without significant changes in hepatocyte cyclic adenosine monophosphate (cAMP) levels and was accompanied by an increase in cytoplasmic concentration of free Ca2+. Parallel to these effects, glibenclamide increased the cellular content of F-2,6-P2, with this effect being associated with a reduction in the rate of glucose formation from a mixture of [14C]lactate/pyruvate. Under similar conditions, meglitinide caused a significant reduction of F-2,6-P2 levels and accelerated the gluconeogenic flux. The mechanism by which meglitinide decreases hepatocyte F-2,6-P2 levels seems to be mediated by stimulation of fructose-2,6-bisphosphatase. This comparative study may help to elucidate which among the hepatic effects of glibenclamide are exerted specifically by the sulfonylurea moiety.

Adenine Nucleotides↗
PubMedDate not supplied

The role of blood platelets in nucleoside metabolism: regulation of platelet thymidine phosphorylase.

Blood platelets are the smallest cellular elements in mammalian blood. Because of their small size, platelets have an unusually large surface area: volume ratio and are exquisitely sensitive to a multitude of physiological and environmental stimuli. Platelets lack nuclei, but most possess functional mitochondria and remain capable of both anaerobic and aerobic energy metabolism, for which they utilise a variety of substrates including many which are cytotoxic and genotoxic for other (nucleated) cells. Nucleic acid precursors are amongst the potentially genotoxic compounds for which platelets have an apparently insatiable appetite. In particular platelets actively scavenge adenine and adenosine, which they convert to nucleotides and use in energy metabolism, but they also rapidly phosphorylase thymidine and liberate thymine into the extracellular medium. In addition, platelets contain non-metabolisable membrane-bound pools of adenine nucleotides which they secrete in response to strong agonists. Taken together, these observations suggest that blood platelets play an important role in nucleic acid precursor metabolism. In the previous paper we have shown that most thymidine phosphorylase activity present in normal human blood resides in the cytoplasm of platelets. Here we demonstrate that this enzyme activity can be modulated in a dose-dependent fashion, not only by substances recognised as platelet agonists and antagonists, but also by some compounds which are considered to be toxic, mutagenic and/or carcinogenic. The data which we present provide additional support for our previous suggestion that platelets regulate thymidine homeostasis and further imply that this is the normal, physiological, platelet function. Preliminary results suggest that assays of blood platelet thymidine metabolism may provide data with a wide variety of applications.

Alcohols↗
PubMed1976

Characterization of a cell culture model for the study of adenosine deaminase- and purine nucleoside phosphorylase-deficient immunologic disease.

The absence of erythrocytic adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) has been associated with severe immunodeficiency disease in children. We have developed a cell culture model to study the possible relationships between purine salvage enzymes and immunologic function using an established T cell lymphosarcoma (S49) and a potent inhibitor of ADA, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA). Wild-type S49 cells are killed by dexamethasone or dbc AMP, and adenosine (5 muM) in the presence of an ADA inhibitor (6 muM EHNA) also prevents the growth of and kills these S49 cells. It has been proposed that adenosine is toxic to lymphoid cells by virtue of its ability to increase the intracellular concentrations of cyclic AMP. We examined the sensitivity of three mutants of S49 cells, with distinctive defects in some component of cyclic AMP metabolism or action, to killing by adenosine and EHNA. All three mutants are resistant to killing by isoproterenol or cholera toxin and two are resistant to dbc AMP itself, but all are sensitive to killing by adenosine and EHNA. Similarly, two dexamethasone-resistant S49 mutants are as sensitive to adenosine and EHNA as are the wildtype cells. We have also simulated the purine nucleoside phosphorylase deficiency in S49 cells by adding inosine and adenosine to the growth medium. In the presence of EHNA or inosine, the toxic effects of adenosine can be partially reversed by addition of (10-20 muM) uridine, an observation suggesting that adenosine is toxic as the result of its inducing pyrimidine starvation.

Adenine↗
PubMed1995

Molecular studies of glycogen phosphorylase b from bovine liver.

Two active isoforms of bovine liver phosphorylase with distinct subunit composition have previously been purified (Cámara Artigas, A., Barón, C. and Parody-Morreale, A. Prot. Express. Purif. 1994, 5, 157), one showing three SDS-PAGE polypeptide bands (molecular mass = 97, 55 and 40 kDa) and the other showing just one (molecular mass = 97 kDa). A molecular mass of 200 kDa has been determined for the native enzymes by gel filtration. Amino acid analyses have been performed in both cases, giving the same results which are similar to those obtained with other phosphorylases. SDS-PAGE experiments at different concentrations of the three-band enzyme have suggested a 1:1:1 stoichiometry between the polypeptides. The pyridoxal-5'-phosphate site is located in the 55 kDa polypeptide and the phosphorylation site in the 40 kDa one. These polypeptides can be generated from the three-band enzyme by tryptic attack in the presence of glycogen without loss of enzyme activity. In the absence of glycogen, 55 kDa and 38 kDa polypeptides are generated, with a significant decrease in activity. We conclude that the three-band enzyme is a dimer composed of an intact monomer and a broken one. The lyotropic salt activation site of the enzyme is near the amino terminal group.

Amino Acids↗
PubMed1990

Localisation of the gene encoding the catalytic gamma subunit of phosphorylase kinase to human chromosome bands 7p12-q21.

Skeletal muscle phosphorylase kinase has the structure (alpha beta gamma delta)4 where the alpha and beta subunits are regulatory components, the gamma subunit possesses catalytic activity and the delta subunit is identical to the calcium binding protein calmodulin. A rabbit skeletal muscle cDNA for the gamma subunit has been used to map the human gene (PYKG1) to 7p12-q21, by analysis of somatic cell hybrids and in situ hybridisation. The data suggest that the skeletal muscle gamma subunit gene is located just above the centromere of chromosome 7, with further cross-hybridising sequences at 7q21 and 11p11-14. The liver gamma subunit is distinct and its mRNA does not cross-hybridize with the skeletal muscle gamma subunit cDNA. These results indicate that autosomal human phosphorylase kinase deficiencies affecting both liver and muscle are likely to be caused by a defect in the autosomally determined beta subunit, rather than the gamma subunit.

Blotting, Northern↗
PubMed1992

Characterization and cloning of glycogen phosphorylase 1 from Dictyostelium discoideum.

We have cloned cDNA and genomic DNA fragments from Dictyostelium discoideum that represent the entire coding region of glycogen phosphorylase 1 (gp1, alpha-D-glucosyltransferase, EC 2.4.1.1). Nucleic acid sequencing of the gp1 clones revealed a single 139 bp intron separating the two exons that encode the 853 amino acids of gp1. The gp1 sequences are similar to other genes and proteins described for Dictyostelium in terms of G + C composition of coding and noncoding regions, splice junctions, intron length, codon preference and termination signals. Genomic Southern blot hybridizations suggest that gp1 exists as a single or low copy number gene in Dictyostelium. Northern analyses demonstrate that gp1 is a developmentally regulated transcript. In alignments of the gp1 peptide sequence to glycogen phosphorylase sequences from other organisms, a high degree of amino acid conservation at many active and regulatory sites was found; however, critical residues in the AMP and purine binding sites were not conserved.

Amino Acid Sequence↗
PubMed1991

Sulphate activates phosphorylase b by binding to the Ser (P) site.

The notion, in a recent crystallographic study on the R state phosphorylase b (Barford, D. and Johnson, L.N. (1989) Nature 340, 609-616), that sulphate ions activate the enzyme by interacting within 2 A of the phosphorylatable Ser-14, is directly supported by kinetic studies on phosphorylase b', a proteolytic species which lacks the N-terminal 16 residues. The results show that this form of the enzyme is no longer activated by ammonium sulphate.

Adenosine Monophosphate↗
PubMed1991

Solution conformations of the N-terminal CNBr fragment of glycogen phosphorylase and its interaction with calmodulin.

The CNBr peptides, CBPa and CBPb, corresponding to the N-terminal 1-91 amino acid residues of glycogen-phosphorylase a and b, respectively, were purified and characterized. CD, 31P-NMR and fluorescence spectroscopy were used to assess the structural organization of the cyanogen bromide peptides in solution. The cyanogen bromide peptides yielded 21% of alpha-helical structures by CD compared to a calculated value of 36.3%. These peptides interact with calmodulin which induces measurable alpha-helices in the cyanogen bromide peptides. The helix stabilizing reagent, trifluoroethanol, induces high numbers of alpha-helices in CBP, thereby demonstrating the conformation fluidity of this peptide. The dissociation constants for calmodulin and CBP estimated by fluorescence titrations were 36.0 and 29.9 nM for CBPb in the presence of Ca2+ and EGTA, respectively. The phosphorylated residue in CBPa causes a decrease in binding interactions with calmodulin and corresponding values obtained for CBPa by fluorescence titration are 56.0 and 141.0 nM, respectively. The Ser-P-14 of CBPa is titratable, yielding a pKa = 5.45 and a Hill coefficient of 1.5. A helical wheel analysis using a computer program in PC/GENE of the CBP shows that peptide stretches in the alpha-1 and alpha-2 helices are most basic and fairly amphiphilic and therefore represent the most probable segment for CaM binding. It is this structural character of these segments which presumably confer the ability to bind CaM and facilitate some of the allosteric transitions of glycogen phosphorylase.

Calcium↗
PubMed1992

Purine nucleoside phosphorylase from bovine lens: purification and properties.

Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was purified 38,750-fold to apparent electrophoretic homogeneity from bovine ocular lens. The enzyme appears to be a homotrimer with a molecular weight of 97,000, and displays non-linear kinetics with concave downward curvature in double-reciprocal plots with orthophosphate as variable substrate. The analysis of the kinetic parameters of bovine lens purine nucleoside phosphorylase, determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases, indicates the occurrence of a rapid equilibrium random Bi-Bi mechanism with formation of abortive complexes. The effect of pH on the enzyme activity and on the sensitivity of the enzyme to photoinactivation, as well as the effect of thiol reagents on the enzyme activity and stability, strongly suggest the involvement of histidine and cysteine residues in the active site. From the measurements of the kinetic parameters at different temperatures, heats of formation of the enzyme-substrate complex for guanosine, guanine, orthophosphate and ribose 1-phosphate were determined. Activation energies of 15,250 and 14,650 cal/mol were obtained for phosphorolysis and synthesis of guanosine, respectively.

Animals↗
PubMed1996

A calorimetric study of the binding of AMP to liver glycogen phosphorylase b.

The energetics of the interaction between liver glycogen phosphorylase b and the adenosine 5'-monophosphate (AMP) have been studied by equilibrium dialysis and isothermal titration calorimetry (ITC) at 25 degrees C. A concomitant net release of protons with AMP to phosphorylase binding was detected carrying out calorimetric experiments in three buffers having different heats of ionization at 25 degrees C. Four binding sites were found for AMP in the dimeric enzyme, which would correspond to the activator and the inhibitor sites identified in the muscle isozyme. The affinity of AMP for these four sites is similar. Thus, the binding of AMP to the activator sites seems to be non-cooperative and it does not perform the conformational change necessary to activate the enzyme. Moreover, the inhibitor sites are occupied almost in the same extension that the activator sites, which would impair any activation of the enzyme.

Adenosine Monophosphate↗
PubMed1985

The turnover of skeletal muscle glycogen phosphorylase studied using the cofactor, pyridoxal phosphate, as a specific label.

The turnover of glycogen phosphorylase has been measured using the cofactor, pyridoxal phosphate, as a label specific for this enzyme in skeletal muscle. Radiolabelled pyridoxine administered in vivo is incorporated into a protein-bound fraction in skeletal muscle, shown by several criteria to be equivalent to glycogen phosphorylase. This pool of radiolabel disappears slowly with a half-life of 11.9 days, taken to be a good estimate of the intracellular half-life of the enzyme. The use of the cofactor in this fashion minimises overestimation of half-life that results from reincorporation of the label. Further, premature dissociation of the cofactor from native enzyme, which would lead to underestimation of half-life, is unlikely. At the level of sensitivity given by this method there was little evidence for the appearance of pyridoxal phosphate-labelled degradation intermediates of the enzyme.

Animals↗
PubMed1985

Differential regulation of polyamine synthesis and transmethylation reactions in methylthioadenosine phosphorylase deficient mammalian cells.

The consumption of S-adenosylmethionine during polyamine synthesis and transmethylation reactions yields stoichiometric amounts of 5'-deoxy-5'-methylthioadenosine and S-adenosylhomocysteine, respectively. Information concerning the regulation of the two routes of S-adenosylmethionine metabolism in viable cells under changing growth conditions is limited. The present experiments have measured the time-dependent accumulation of 5'-deoxy-5'-methylthioadenosine and L-homocysteine in the medium of four malignant human and murine cell lines deficient in 5'-deoxy-5'-methylthioadenosine phosphorylase (5'-methylthioadenosine: orthophosphate methylthioribosyltransferase). Included in this group were anchorage-independent and anchorage-dependent cells. The enzyme-deficient cells did not detectably cleave 5'-deoxy-5'-methylthioadenosine, and did not appreciably metabolize homocysteine. A comparison of 5'-deoxy-5'-methylthioadenosine and homocysteine excretion therefore provided a noninvasive method for estimating the relative rates of polyamine synthesis and transmethylation. Early after the release of human CEM lymphoblasts from density dependent growth arrest, 5'-deoxy-5'-methylthioadenosine production increased, and exceeded homocysteine synthesis. 5'-Deoxy-5'-methylthioadenosine formation reached a maximum of 0.9 nmol/12 h per 10(6) cells prior to the onset of exponential growth. The kinetics of homocysteine synthesis were different. Homocysteine accumulation was proportional to the specific growth rate, and achieved a peak of 3.1 nmol/12 h per 10(6) cells during mid-exponential phase, at which time 5'-deoxy-5'-methylthioadenosine production was falling. Similar patterns of 5'-deoxy-5'-methylthioadenosine and homocysteine excretion were observed in other 5'-deoxy-5'-methylthioadenosine phosphorylase deficient cell lines. These data show that polyamine synthesis and transmethylation are differentially regulated during the growth cycle of mammalian cells.

Adenosine↗
PubMed1987

Synthesis of purines in human lymphoblast cells deficient in methylthioadenosine phosphorylase activity.

Two human lymphoblastic cell lines, deficient in methylthioadenosine phosphorylase (MTAP) activity, were found to have increased rates of de novo purine synthesis. These MTAP- cell lines were K562, an undifferentiated leukemic line and CCRF-CEM, a leukemic line of T-cell origin. Another T-cell line, CCRF-HSB-2 was found to be deficient in activity. However, this line did not demonstrate elevated rates of purine synthesis. Purine metabolism in the above cell cultures was compared with MTAP+ human B-cell lines and two human T-cell lines (MOLT-3 and MOLT-4). In all the MTAP+ cell lines, the rate of de novo purine synthesis was inhibited by the presence of methylthioadenosine in the assay medium (10 microM concentration produced more than 90% inhibition). However, purine synthesis in the MTAP- cells was resistant to inhibition by methylthioadenosine. Adenine in the assay medium inhibited de novo purine synthesis in MTAP+ and MTAP- cells to a similar degree. This inhibition was dose dependent and was elicited by concentrations similar to those of methylthioadenosine. Growth of the cell lines in culture was not affected by either methylthioadenosine or adenine at the concentrations which produced inhibition of purine synthesis. These results suggest that purine synthesis in MTAP+ cells is inhibited by adenine formed from the phosphorolytic cleavage of methylthioadenosine by methylthioadenosine phosphorylase.

Adenine↗