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PubMed1983

Glycogen metabolism in the capillary endothelium. Electron histochemical study of glycogen synthetase and phosphorylase in the pecten capillary of the chick.

Glycogen synthetase and phosphorylase activities in the pecten capillary of the chick were studied electron histochemically. Polyglucose particles synthesized by glycogen synthetase or phosphorylase in the pecten capillary were demonstrated to be amorphous. They were located in the cytoplasmic matrix of the endothelium and expanded it widely. The endothelial cell of the pecten capillary plays a role in glycogenesis as well as glycogenolysis and may require substantial energy to its important transport functions.

Animals↗

PubMed1988

Comparison of the pyrimidine nucleoside phosphorylase activity in human tumors and normal tissues.

The novel fluoropyrimidine, 5'-deoxy-5-fluorouridine (5'-dFUDR) has strong tumor-inhibiting effect without severe cytotoxic effects on the normal cells. This pro-drug is metabolized by the enzyme pyrimidine nucleoside phosphorylase (PN'ase) to the active form 5-fluorouracil. Comparative determinations of pyrimidine nucleoside phosphorylase activity in tissue extracts revealed a significantly higher activity in the human gastrointestinal cancer tissues (6.84 +/- 0.70 nmol/mg protein) than in normal tissues from the same organ (2.37 +/- 0.21 nmol/min/mg protein).

Colonic Neoplasms↗

PubMed1977

Multiple molecular forms of phosphorylase phosphatase associated with particulate glycogen and extracted from the cytosol of dog liver.

The glycogen pellet of dog liver extracts contains a phosphorylase phosphatase which has characteristics different from those of the phosphatases extracted from the cytosol. The phosphatase associated with glycogen is characterized by a M, of 51,000, a half maximal inhibition at 0.3 mM ATP (Hill coefficient : 2) and a Ki for Mg2+ of 1 mM. Treatment with urea or mercaptoethanol of the phosphatase associated with glycogen does not influence the activity, the Mr or the half maximal inhibition by ATP, but a decrease of the Hill coefficient for ATP is observed. A similar treatment of the phosphatases extracted from the high speed supernatant results in a decrease of the Mr of the spontaneously active form from 215,000 to 43,000, without an effect on the Ki for ATP (7 micronM), but accompanied by an increase in activity. The ATP-Mg dependent form of the phosphatase from the high speed supernatant (Mr : 138,000 ; Ka for ATP in the presence of 0.1 mM Mg2+ : 0.3 micronM), is denatured by urea or mercaptoethanol. The phosphatase associated with particulate glycogen cannot be found in the supernatant, nor the phosphorylase phosphatases present in the supernatant in the glycogen pellet. When all the glycogen is mobilized (starvation, glucagon) the phosphatase specifically associated with glycogen cannot be found as such in the cytosol. No activation of synthase beta can be detected neither with the phosphatases extracted from the cytosol nor with the enzyme released from the glycogen pellet.

Adenosine Triphosphate↗

PubMed2001

Combined kinetic mechanism describing activation and inhibition of muscle glycogen phosphorylase b by adenosine 5'-monophosphate.

The kinetic analysis of the glycogen chain growth reaction catalyzed by glycogen phosphorylase b from rabbit skeletal muscle has been carried out over a wide range of concentrations of AMP under the saturation of the enzyme by glycogen. The applicability of 23 different variants of the kinetic model involving the interaction of AMP and glucose 1-phosphate binding sites in the dimeric enzyme molecule is considered. A kinetic model has been proposed which assumes: (i) the independent binding of one molecule of glucose 1-phosphate in the catalytic site on the one hand, and AMP in both allosteric effector sites and both nucleoside inhibitor sites of the dimeric enzyme molecule bound by glycogen on the other hand; (ii) the binding of AMP in one of the allosteric effector sites results in an increase in the affinity of other allosteric effector site to AMP; (iii) the independent binding of AMP to the nucleoside inhibitor sites of the dimeric enzyme molecule; (iv) the exclusive binding of the second molecule of glucose 1-phosphate in the catalytic site of glycogen phosphorylase b containing two molecules of AMP occupying both allosteric effector sites; and (v) the catalytic act occurs exclusively in the complex of the enzyme with glycogen, two molecules of AMP occupying both allosteric effector sites, and two molecules of glucose 1-phosphate occupying both catalytic sites.

Adenosine Monophosphate↗

PubMed1998

Irreversible thermal denaturation of uridine phosphorylase from Escherichia coli K-12.

Thermal denaturation of uridine phosphorylase from Escherichia coli K-12 has been studied by differential scanning calorimetry. The excess heat capacity vs. temperature profiles were obtained at temperature scanning rates of 0.25, 0.5, and 1 K/min. These profiles were analysed using three models of irreversible denaturation which are approximations to the whole Lumry-Eyring model, namely, the one-step model of irreversible denaturation, the Lumry-Eyring model with the fast equilibrating first step, and the model involving two consecutive irreversible steps. In terms of statistics the latter model describes the kinetics of thermal denaturation of uridine phosphorylase more satisfactorily than the two other models. The values of energy activation for the first and second steps calculated for the model involving two consecutive irreversible steps are the following: Ea,1 = 609.3 +/- 1.8 kJ/mol and Ea,2 = 446.8 +/- 3.2 kJ/mol.

Calorimetry, Differential Scanning↗

PubMed1987

Adenosine deaminase and purine phosphorylase activities in lymphocytes and red blood cells of patients with carcinoma of the larynx.

Lymphocytes from patients with cancer of the larynx have been characterized by the activity of two enzymes involved in purine metabolism--adenosine deaminase and purine nucleoside phosphorylase. It has been shown that purine nucleoside phosphorylase activity was 1.5-fold higher in lymphocytes of patients in comparison with a group of healthy individuals. Adenosine deaminase activity in lymphocytes was elevated three to four-fold. The authors suggest that especially the estimation of adenosine deaminase activity in lymphocytes would be a useful laboratory test in the study of immune responsiveness of patients with cancer of the larynx.

Adenosine Deaminase↗

PubMed2003

Effects of chronic ethanol treatment on the angiotensin II-mediated p42/p44 mitogen-activated protein kinase and phosphorylase a activation in rat hepatocytes.

We have demonstrated previously that 24 h of ethanol treatment potentiates angiotensin II (ANG II)-stimulated p42/p44 mitogen-activated protein kinase (MAPK) activity in hepatocytes. This potentiation of p42/p44 MAPK by ethanol exhibited agonist selectivity. To compare the effects of acute (24 h) versus chronic (6 weeks) ethanol treatment, ANG II-induced intracellular signaling was examined in (1) rat hepatocytes treated with ethanol for 24 h and (2) hepatocytes obtained from rats fed ethanol for 6 weeks. In hepatocytes obtained from rats fed ethanol for 6 weeks, ANG II-stimulated phosphorylase a was reduced, and this activity was calcium dependent and p42/p44 MAPK independent. Surprisingly, ANG II-stimulated p42/p44 MAPK activation was not affected in hepatocytes obtained from rats fed ethanol chronically (6 weeks). However, chronic (6 weeks) ethanol treatment decreased ethanol potentiation of p42/p44 MAPK by about 56.3% +/- 3.6% for p42 MAPK and 61.3% +/- 11.7% for p44 MAPK. Furthermore, ethanol had no effect on the expression of angiotensinogen and c-myc mRNA in hepatocytes. A decrease in ANG II-activated phosphorylase a, but not in p42/p44 MAPK activation, after chronic (6 weeks) ethanol treatment leads to the conclusion that they may not be dependent on each other.

Angiotensin II↗

PubMedDate not supplied

Adenosine phosphorylase activity as a technique for detection of mycoplasmas in biological media.

The importance of cell culture contamination by mycoplasmas is well recognized, but the means used to detect such contamination still need improvement. Most mycoplasmas possess an enzyme, adenosine phosphorylase, which is not found in cell lines. We used the ultramicromethod of Uitendaal et al. to detect the presence of mycoplasmas in sera and in tissue culture medium. The absence of adenosine phosphorylase activity seems to be the best guarantee that a serum is not contaminated by mycoplasmas. This test is also most efficient for the detection of mycoplasmas in tissue or cell cultures in vitro.

Animals↗

PubMed2003

Polyethylene glycol-conjugated adenosine phosphorylase: development of alternative enzyme therapy for adenosine deaminase deficiency.

Purine nucleoside phosphorylase had previously been engineered to accept 6-amino substituted purine nucleosides by two active site substitutions, Asn243Asp; Lys244Gln. In the present study, recombinant adenosine phosphorylase (AP) has been conjugated to branched polyethylene glycol (PEG) polymers of approximately 42.5 kDa. Matrix-assisted laser desorption/ionization analysis and SDS acrylamide electrophoresis analysis indicated a subunit composition of greater than 205 kDa consistent with the conjugation of as many as four PEG molecules per AP subunit. The PEG-conjugated enzyme retained greater than 90% of the native catalytic activity. Administration of the enzyme to mice demonstrated the PEG-AP to have a 67-fold increased plasma half-life compared to the native enzyme, 65.1+/-2.9 h versus 57.8+/-1.1 min, respectively. PEG-AP was principally confined to the plasma with minimal activity detected in tissues and of these spleen had the greatest activity and essentially no activity was found in urine. PEG-AP has retained activity with inosine and its injection into PNP-deficient mice resulted in a 2.7-fold increase in urine urate. AP was also shown to protect human CEM cells in culture from the toxic effects of 2'-deoxyadenosine. These studies provide evidence for consideration of PEG-AP as an alternative enzyme therapy for the inherited deficiency of adenosine deaminase.

Adenosine Deaminase↗

PubMed1997

Further syntheses employing phosphorylase.

Maltopentaose was immobilized on silica gel and used as a recyclable primer in the reaction of glycogen phosphorylase with D-glucal. An improved method to obtain 2-deoxy-alpha-D-arabino-hexopyranosyl phosphate (4) by using this catalyst as well as the specific synthesis of low molecular weight, water-soluble 2-deoxy-maltooligosaccharides (12, 13, 14 and 15) are described. Further investigations with modified phosphorylase substrates showed that mannosyl phosphate (16) can be slowly transferred to the primer maltotetraose. alpha-1,4-Mannosyl-maltotetraose (17) and its degradation product alpha-1,4-mannosyl-maltose (18) were identified.

Carbohydrate Sequence↗

PubMed1997

The structure of glycogen phosphorylase b with an alkyldihydropyridine-dicarboxylic acid compound, a novel and potent inhibitor.

BACKGROUND: In muscle and liver, glycogen concentrations are regulated by the reciprocal activities of glycogen phosphorylase (GP) and glycogen synthase. An alkyl-dihydropyridine-dicarboxylic acid has been found to be a potent inhibitor of GP, and as such has potential to contribute to the regulation of glycogen metabolism in the non-insulin-dependent diabetes diseased state. The inhibitor has no structural similarity to the natural regulators of GP. We have carried out structural studies in order to elucidate the mechanism of inhibition. RESULTS: Kinetic studies with rabbit muscle glycogen phosphorylase b (GPb) show that the compound (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5, 6-tricarboxylate (Bay W1807) has a Ki = 1.6 nM and is a competitive inhibitor with respect to AMP. The structure of the cocrystallised GPb-W1807 complex has been determined at 100K to 2.3 A resolution and refined to an R factor of 0.198 (Rfree = 0.287). W1807 binds at the GPb allosteric effector site, the site which binds AMP, glucose-6-phosphate and a number of other phosphorylated ligands, and induces conformational changes that are characteristic of those observed with the naturally occurring allosteric inhibitor, glucose-6-phosphate. The dihydropyridine-5,6-dicarboxylate groups mimic the phosphate group of ligands that bind to the allosteric site and contact three arginine residues. CONCLUSIONS: The high affinity of W1807 for GP appears to arise from the numerous nonpolar interactions made between the ligand and the protein. Its potency as an inhibitor results from the induced conformational changes that lock the enzyme in a conformation known as the T' state. Allosteric enzymes, such as GP, offer a new strategy for structure-based drug design in which the allosteric site can be exploited. The results reported here may have important implications in the design of new therapeutic compounds.

Adenosine Monophosphate↗

PubMed1998

The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation.

BACKGROUND: Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide synthesis salvage pathway. In lower organisms (e.g. Bacillus stearothermophilus) PYNP accepts both thymidine and uridine, whereas in mammalian and other higher organisms it is specific for thymidine (designated thymidine phosphorylase, TP). PYNP shares 40% sequence similarity (and presumably significant structural similarity) with human TP, which has been implicated as a growth factor in tumor angiogenesis. It is thought that TP undergoes a major conformational change upon substrate binding that consequently produces an active conformation. RESULTS: The crystal structure of PYNP from B. stearothermophilus with the substrate analog pseudouridine in its active site has been solved to 2.1 A resolution. This structure confirms the similarity of PYNP to TP and supports the idea of a closed active conformation, which is the result of rigid body movement of the alpha and alpha/beta domains. The active-site cleft, where the pyrimidine and phosphate substrates bind, is between the two domains. The structure reveals an asymmetric dimer in which one subunit is fully closed and the other is only partially closed. CONCLUSIONS: The closed conformation of PYNP serves as a good model to better understand the domain movement and overall function of TP. Active-site residues are confirmed and a possible mechanism for substrate binding and subsequent domain movement is suggested. Potent inhibitors of TP might have significant therapeutic value in various chemotherapeutic strategies, and the structure of PYNP should provide valuable insight into the rational design of such inhibitors.

Binding Sites↗

PubMed1999

The structure of human 5'-deoxy-5'-methylthioadenosine phosphorylase at 1.7 A resolution provides insights into substrate binding and catalysis.

BACKGROUND: 5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAP) catalyzes the reversible phosphorolysis of 5'-deoxy-5'-methylthioadenosine (MTA) to adenine and 5-methylthio-D-ribose-1-phosphate. MTA is a by-product of polyamine biosynthesis, which is essential for cell growth and proliferation. This salvage reaction is the principle source of free adenine in human cells. Because of its importance in coupling the purine salvage pathway to polyamine biosynthesis MTAP is a potential chemotherapeutic target. RESULTS: We have determined the crystal structure of MTAP at 1.7 A resolution using multiwavelength anomalous diffraction phasing techniques. MTAP is a trimer comprised of three identical subunits. Each subunit consists of a single alpha/beta domain containing a central eight-stranded mixed beta sheet, a smaller five-stranded mixed beta sheet and six alpha helices. The native structure revealed the presence of an adenine molecule in the purine-binding site. The structure of MTAP with methylthioadenosine and sulfate ion soaked into the active site was also determined using diffraction data to 1.7 A resolution. CONCLUSIONS: The overall quaternary structure and subunit topology of MTAP are similar to mammalian purine nucleoside phosphorylase (PNP). The structures of the MTAP-ligand complexes provide a map of the active site and suggest possible roles for specific residues in substrate binding and catalysis. Residues accounting for the differences in substrate specificity between MTAP and PNP are also identified. Detailed information about the structure and chemical nature of the MTAP active site will aid in the rational design of inhibitors of this potential chemotherapeutic target. The MTAP structure represents the first structure of a mammalian PNP that is specific for 6-aminopurines.

Amino Acid Sequence↗

PubMed2003

cDNA microarray analysis of gene expression profiles in human placenta: up-regulation of the transcript encoding muscle subunit of glycogen phosphorylase in preeclampsia.

OBJECTIVE: Third-trimester human placentas from normal and preeclamptic pregnancies were evaluated for possible changes in gene expression patterns by microarray analysis. METHODS: Placentas from four normal pregnancies and four pregnancies complicated by preeclampsia were studied. In a preliminary effort to identify possible differences between the two groups, complementary DNA (cDNA) probes were prepared from pooled total RNA by reverse transcription in the presence of (33)P-dCTP. After hybridization to human GeneFilter cDNA microarrays (GF211; Research Genetics, Huntsville, AL), 319 positive signals were detected above background out of a possible 4131 human cDNAs spotted on the filters. RESULTS: Ten most highly expressed mRNA species, ten most up-regulated, and ten most down-regulated genes in placentas from both groups of patients were identified for future studies. Of the 319 positive hybridizations, one transcript was clearly elevated in preeclamptic pregnancy. This cDNA encodes the muscle subtype of glycogen phosphorylase (GP-M) and was increased more than 2.8-fold (P <.05) in the preeclamptic placentas. In contrast, cDNA for glycogen synthase (muscle and liver isoforms) was not significantly increased, being near the limits of detection. The preeclampsia-induced increase of placental GP-M mRNA expression (approximately 3.5-fold) was confirmed by northern blot analysis. CONCLUSION: We conclude that microarray analysis can detect trends in mRNA and gene expression in placentas from normal and preeclamptic pregnancies that may be further studied in a more targeted fashion. We found that placental GP-M mRNA level is up-regulated in preeclampsia, which is consistent with previous reports of increased glycogen phosphorylase activity, and we suggest that it may be largely regulated at the level of transcription. Further studies may determine whether such up-regulation might be a response to hypoxia.

Female↗

PubMed1995

Pre-steady-state transition-state analysis of the hydrolytic reaction catalyzed by purine nucleoside phosphorylase.

The slow hydrolytic reaction catalyzed by calf spleen purine nucleoside phosphorylase [Kline, P. C., & Schramm, V. L. (1992) Biochemistry 31, 5964-5973] has been investigated using pre-steady-state kinetic isotope effects and solvolysis studies. The stoichiometric reaction between enzyme and inosine forms 1 mol of free ribose per trimer of purine nucleoside phosphorylase and a tightly bound complex of enzyme and hypoxanthine. The experimental kinetic isotope effects from [1'-3H]-, [2'-3H]-, [4'-3H]-, [5'-3H]-, [1'-14C]-, and [9-15N]inosine are 1.151 +/- 0.004, 1.145 +/- 0.003, 1.006 +/- 0.004, 1.028 +/- 0.005, 1.045 +/- 0.005, and 1.000 +/- 0.005, respectively, for the pre-steady-state conditions. Substrate trapping experiments demonstrated that there is no detectable forward commitment to catalysis for inosine hydrolysis. In contrast, bound inosine is 2.1 times more likely to form product than to dissociate when the enzyme-inosine complex is exposed to saturating PO4. The lack of an observed 9-15N isotope effect is consistent with an internal equilibrium between enzyme-inosine and the enzyme-hypoxanthine-ribose complex in which N9 of hypoxanthine is protonated. The equilibrium occurs as a consequence of slow product release and tightly bound hypoxanthine (Kd = 1.3 x 10(-12) M). This internal equilibrium has a minimal effect on the intrinsic kinetic isotope effects from ribose since equilibrium isotope effects for conversion of inosine to ribose are near unity. When the single-turnover hydrolytic reaction was accomplished in 20% methanol, approximately 85% of the product sugar was 1-methylribose. Under these conditions, the anion-binding pocket fills with solvent which competes for the oxocarbenium ion of inosine formed at the transition state. In the presence of arsenate, no methanolysis of inosine occurs [Kline, P. C., & Schramm, V. L. (1993) Biochemistry 32, 13212-13219]. The results define a transition state with oxocarbenium ion character and weak participation of the attacking solvent nucleophile. Electrostatic potential surfaces of the transition states indicate that arsenate anion is more effective in neutralizing the oxocarbenium ion than is H2O.

Adenosine↗

PubMed1993

Physical heterogeneity of muscle glycogen phosphorylase revealed by hydrostatic pressure dissociation.

Four independent methods that employ fluorescence spectroscopy show that the tetramer of glycogen phosphorylase A (GPA) from rabbit muscle is reversibly dissociated into monomers by hydrostatic pressures under 2.5 kbar, if aggregation of the monomers is prevented by the addition of 8% glycerol. The free energy of association at 20 degrees C (-32 kcal mol-1) depends upon a large entropy increase (T delta S = +65 kcal mol-1) that counteracts an unfavorable enthalpy of association of +33 kcal mol-1. The association volumes calculated from the pressure dependence of the dissociation are nearly 4-fold smaller than those calculated from the shift in dissociation pressure with concentration. The dimer obtained by dilution of GPA at atmospheric pressure differs from the hypothesized dimer intermediate in the pressure dissociation by the much larger monomer affinity of the former. Like other tetramers, GPA shows hysteresis of the pressure profile upon decompression and conformational drift of the dissociated monomers. By use of the energy transfer method it is demonstrated that the relaxation time for half-dissociation (5 min) is over an order of magnitude shorter than that for subunit exchange (118 min). In all three tetramers studied, lactate dehydrogenase, glyceraldehyde phosphate dehydrogenase, and glycogen phosphorylase, the deterministic character of the dissociation equilibrium under pressure and the anomalous concentration dependence of the pressure dissociation demonstrate that these tetramers are heterogeneous populations with regard to their free energy and/or volumes of association.

Animals↗

PubMed1991

Glucose analogue inhibitors of glycogen phosphorylase: the design of potential drugs for diabetes.

The T-state crystal structure of the glucose-phosphorylase b complex has been used as a model for the design of glucose analogue inhibitors that may be effective in the regulation of blood glucose levels. Modeling studies indicated room for additional atoms attached at the C1-beta position of glucose and some scope for additional atoms at the C1-alpha position. Kinetic parameters were determined for alpha-D-glucose: Ki = 1.7 mM, Hill coefficient n = 1.5, and alpha (synergism with caffeine) = 0.2. For beta-D-glucose, Ki = 7.4 mM, n = 1.5, and alpha = 0.4. More than 20 glucose analogues have been synthesized and tested in kinetic experiments. Most were less effective inhibitors than glucose itself and the best inhibitor was alpha-hydroxymethyl-1-deoxy-D-glucose (Ki = 1.5 mM, n = 1.3, alpha = 0.4). The binding of 14 glucose analogues to glycogen phosphorylase b in the crystal has been studied at 2.4-A resolution and the structure have been refined to crystallographic R values of less than 0.20. The kinetic and crystallographic studies have been combined to provide rationalizations for the apparent affinities of glucose and the analogues. The results show the discrimination against beta-D-glucose in favor of alpha-D-glucose is achieved by an additional hydrogen bond made in the alpha-glucose complex through water to a protein group and an unfavorable environment for a polar group in the beta pocket. The compound alpha-hydroxymethyl-1-deoxy-D-glucose has an affinity similar to that of glucose and makes a direct hydrogen bond to a protein group. Comparison of analogues with substituent atoms that have flexible geometry (e.g., 1-hydroxyethyl beta-D-glucoside) with those whose substituent atoms are more rigid (e.g., beta-azidomethyl-1-deoxyglucose or beta-cyanomethyl-1-deoxyglucose) indicates that although all three compounds make similar polar interactions with the enzyme, those with more rigid substituent groups are better inhibitors. In another example, alpha-azidomethyl-1-deoxyglucose was a poor inhibitor. In the crystal structure the compound made several favorable interactions with the enzyme but bound in an unfavorable conformation, thus providing an explanation for its poor inhibition. Attempts to utilize a contact to a buried aspartate group were partially successful for a number of compounds (beta-aminoethyl, beta-mesylate, and beta-azidomethyl analogues). The beta pocket was shown to bind gentiobiose (6-O-beta-D-glucopyranosyl-D-glucose), indicating scope for binding of larger side groups for future studies.

Deoxyglucose↗

PubMed1992

Solution structure of phosphorylase kinase studied using small-angle X-ray and neutron scattering.

Small-angle X-ray and neutron scattering have been used to characterize the solution structure of rabbit skeletal phosphorylase kinase. The radius of gyration of the unactivated holoenzyme determined from neutron scattering is 94 A, and its maximum dimension is approximately 275-295 A. A planar model has been constructed that is in general agreement with the dimensions of the transmission electron microscope images of negatively stained phosphorylase kinase and that gives values for the radius of gyration, maximum linear dimension, and a pair distribution function for the structure that are consistent with the scattering data.

Animals↗