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PubMed1986

Activation of phosphorylase kinase through autophosphorylation by membrane component phospholipids.

Phosphatidic acid (PtdOH) has been shown not only to stimulate autophosphorylation and autoactivation of phosphorylase kinase of rabbit skeletal muscle but also to decrease the apparent Ka for Ca2+ on autophosphorylation sharply [Negami et al. (1985) Biochem. Biophys. Res. Commun. 131, 712-719]. In this study we investigated the interaction between PtdOH and other phospholipids on autophosphorylation and autoactivation of this enzyme. Acidic phospholipids, such as phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns) and PtdOH, stimulated this reaction about 2-4-fold, and the approximate Ka values of this reaction were 10 micrograms/ml, 6.3 micrograms/ml and 30 micrograms/ml respectively. The molar ratio of PtdIns and PtdSer with maximal effect on autophosphorylation was about 1:1. Under these conditions PtdOH stimulated the initial velocity of autophosphorylation about 5.2-fold. When fully autophosphorylated, about 12-13 mol phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of mixed acidic phospholipids (PtdOH:PtdIns:PtdSer = 2:1:1), which was about twice as much as values observed without effectors. In the presence of mixed acidic phospholipids there was a concomitant enhancement of kinase activity, about 30-40-fold at pH 6.8 and 2.5-3-fold at pH 8.2. Mixed acidic phospholipids sharply decreased an apparent Ka for Ca2+ from 4 X 10(-5) M to 8 X 10(-7) M. With mixed acidic phospholipids as effectors this autophosphorylation occurred through an intramolecular mechanism. Based on these results, autophosphorylation and autoactivation of phosphorylase kinase in the presence of acidic phospholipids may account for an important regulatory mechanism of glycogenolysis in muscle contraction.

Adenosine Triphosphate↗
PubMed1988

Uridine(5')diphospho(1)-alpha-D-glucose. A binding study to glycogen phosphorylase b in the crystal.

UDP-glucose is an R-state inhibitor of glycogen phosphorylase b, competitive with the substrate, glucose 1-phosphate and noncompetitive with the allosteric activator, AMP. Diffusion of 100 mM UDP-glucose into crystals of phosphorylase b resulted in a difference Fourier synthesis at 0.3-nm resolution that showed two peaks: (a) binding at the allosteric site and (b) binding at the catalytic site. At the allosteric site the whole of the UDP-glucose molecule can be located. It is in a well defined folded conformation with its uracil portion in a similar position to that observed for the adenine of AMP. The uracil and the glucose moieties stack against the aromatic side chains of Tyr-75 and Phe-196, respectively. The phosphates of the pyrophosphate component interact with Arg-242, Arg-309 and Arg-310. At the catalytic site, the glucose-1-P component of UDP-glucose is firmly bound in a position similar to that observed for glucose 1-phosphate. The pyrophosphate is also well located with the glucose phosphate interacting with the main-chain NH groups at the start of the glycine-loop alpha helix and the uridine phosphate interacting through a water molecule with the 5'-phosphate of the cofactor pyridoxal phosphate and with the side chains of residues Tyr-573, Lys-574 and probably Arg-569. However the position of the uridine cannot be located although analysis by thin-layer chromatography showed that no degradation had taken place. Binding of UDP-glucose to the catalytic site promotes extensive conformational changes. The loop 279-288 which links the catalytic site to the nucleoside inhibitor site is displaced and becomes mobile. Concomitant movements of residues His-571, Arg-569, and the loop 378-383, together with the major loop displacement, result in an open channel to the catalytic site. Comparison with other structural results shows that these changes form an essential feature of the T to R transition. They allow formation of the phosphate recognition site at the catalytic site and destroy the nucleoside inhibitor site. Kinetic experiments demonstrate that UDP-glucose activates the enzyme in the presence of high concentrations of the weak activator IMP, because of its ability to decrease the affinity of IMP for the inhibitor site.

Allosteric Site↗
PubMed1990

Localization of phosphoserine residues in the alpha subunit of rabbit skeletal muscle phosphorylase kinase.

The alpha subunit of skeletal muscle phosphorylase kinase, as isolated, carries phosphate at the serine residues 1018, 1020 and 1023. Employing the S-ethyl-cysteine method, these residues are found to be phosphorylated partially, i.e. differently phosphorylated species exist in muscle. Serine 1018 is a site which can be phosphorylated by the cyclic-AMP-dependent protein kinase. The serine residues 972, 985 and 1007 are phosphorylated by phosphorylase kinase itself when its activity is stimulated by micromolar concentrations of Ca2+. These phosphorylation sites are not identical to those found to be phosphorylated already in the enzyme as prepared from freshly excised muscle. A 'multiphosphorylation loop' uniquely present in this but not in the homologous beta subunit contains all the phosphoserine residues so far identified in the alpha subunit.

Amino Acid Sequence↗
PubMed1993

Thymidine phosphorylase activity of platelet-derived endothelial cell growth factor is responsible for endothelial cell mitogenicity.

Recombinant human platelet-derived endothelial cell growth factor, expressed in the yeast Saccharomyces cerevisiae was purified to greater than 98% purity by anion-exchange and hydroxyapatite chromatography. It was shown to possess thymidine phosphorolytic activity in vitro (pH optimum, pH 5.3; Km, 0.11 mM; Vmax, 12.5 mmol min-1 mg-1; turnover number, 9.4 s-1). Covalent modification simultaneously inhibited the enzymatic and mitogenic properties of the protein, while interaction with a cell-surface receptor was not required to stimulate mitogenesis. Purified Escherichia coli thymidine phosphorylase was also mitogenic toward endothelial cells. It is proposed that platelet-derived endothelial cell growth factor is human thymidine phosphorylase which promotes endothelial cell proliferation by reducing thymidine levels that would otherwise be inhibitory to endothelial cell growth.

Cells, Cultured↗
PubMed1995

The gamma subunit of phosphorylase kinase contains a pseudosubstrate sequence.

The catalytic subunit, gamma, of phosphorylase kinase is regulated by a complex set of interactions involving the calcium-binding protein calmodulin and two other subunits designated alpha and beta. These interactions regulate gamma activity that, at least for the calmodulin interactions, involves the regulatory domain in gamma spanning residues 302-366. Within this regulatory domain, we report the identification of a sequence (residues 326-334) that resembles the phosphorylation site in gamma substrates with the exception that a V residue (V332) occurs at the analogous position of the phosphorylated S/T residue. The inhibitory properties of the sequence were assayed with a 10-amino-acid peptide of the sequence. This peptide inhibits a truncated version of gamma, residues 1-300, which is missing the regulatory domain, more potently than it inhibits full-length gamma, and it is a better inhibitor of the full-length gamma at pH 8.2 than at pH 6.8. A similar peptide of the same sequence, except for a S substitution of the V residue, is a good substrate with a comparable Km and better Vmax than peptides of similar length that represent the phosphorylation site in the substrate of the enzyme, glycogen phosphorylase. A mutant gamma protein, with a S for V332 substitution ([V332S]gamma), was prepared using the baculovirus expression system. [V332S]gamma autophosphorylates by an intramolecular mechanism. This demonstrates that this sequence can occupy the catalytic site in the protein. Development of [V332S]gamma affords an experimental model in which the effects of the regulatory factors on autophosphorylation can be determined.

Amino Acid Sequence↗
PubMed1995

2-Chloro-2'-deoxyadenosine (cladribine) and its analogues are good substrates and potent selective inhibitors of Escherichia coli purine-nucleoside phosphorylase.

2-Chloro-2'-deoxyadenosine (CldAdo), a nucleoside that has proven useful in the treatment of several chronic lymphoid malignancies, and its analogue, 2-bromo-2'-deoxyadenosine, are both effective inhibitors of the bacterial (Escherichia coli) purine-nucleoside phosphorylase (PNP), with Ki values of 4.5 microM and 6.3 microM, respectively. The examination of a series of base-modified analogues of CldAdo has shown that several other compounds have similar inhibitor properties, and has indicated that 6-benzyloxy-2-chloro-9-(2'-deoxy-beta-D-ribofuranosyl)purine is the most potent inhibitor with a Ki value of 0.5 microM, competitive with respect to inosine (Ino). CldAdo itself and its base-modified analogues, discounting those substituted at C(8), are also substrates for the E. coli PNP and undergo rapid glycosidic bond cleavage. CldAdo is degraded with substrate efficiency, i.e. Vmax/Km similar to that observed for Ino (130%), although the individual kinetic constants, Km and Vmax, are both approximately an order of magnitude lower than for Ino. All compounds tested are totally inactive as substrates and inhibitors for mammalian (calf spleen) PNP and therefore constitute a new class of potent selective, although cleavable, inhibitors of bacterial phosphorylases. 8-Bromo-2-chloro-2'-deoxyadenosine and 8-thio-2-chloro-2'-deoxyadenosine are the only base-modified CldAdo derivatives showing inhibitory activity against MOLT-3 (acute T-cell leukemia) and U-937 (histiocytic lymphoma) cells and, as shown in this study, are resistant to degradation by E. coli PNP. The above-mentioned results suggest that both analogues could be effective as oral cytotoxic agents that are noncleavable by enteric bacteria.

Cladribine↗
PubMed1990

Phosphorylase alpha and labile metabolites during anoxia: correlation to membrane fluxes of K+ and Ca2+.

The objective of the present study was to explore mechanisms responsible for activation of ion conductances in the initial phases of brain ischemia, particularly for the early release of K+ that precedes massive cell depolarization, and rapid downhill fluxes of K+, Na+, Cl-, and Ca2+. As it has been speculated that a K+ conductance can be activated either by an increase in the free cytosolic calcium concentration (Ca2+i) or by a fall in ATP concentration, the question arises whether the early increase in extracellular K+ concentration (K+e) is preceded by a rise in Ca2+i and/or a fall in ATP content. In the present experiments, ischemia was induced in rats by cardiac arrest, the time courses of the rise in K+e and cellular depolarization were determined by microelectrodes, and the tissue was frozen in situ through the exposed dura for measurements of levels of labile metabolites. including adenine nucleotides and cyclic AMP (cAMP), after ischemic periods of 15, 30, 60, and 120 s. Conversion of phosphorylase b to a was assessed, because it depends, among other things, on changes in Ca2+i. The K+e value rose within a few seconds following induction of ischemia, but massive depolarization (which is accompanied by influx of calcium) did not occur until after approximately 65 s. Activation of phosphorylase was observed already after 15 s and before glycogenolysis had begun. At that time, 3',5'-cAMP concentrations were unchanged, and total 5'-AMP concentrations were only moderately increased. The results demonstrate that a K+ conductance is activated at a time when the overall ATP concentration remains at 95% of control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

Effect of acetylcholine on changes in contractility, heart rate and phosphorylase activity produced by isoprenaline, salbutamol and amino-phylline in the perfused guinea-pig heart.

1. Isolated perfused hearts of guinea-pigs were given graded doses of isoprenaline, salbutamol and aminophylline, both before and during acetylcholine infusion. 2. The three agonists produced increases in contractile force, heart rate and ventricular glycogen phosphorylase activity. 3. Acetylcholine, in the concentration used, had no effect on any of the measured variables but did antagonize the effects of the three agonists on contractility and phosphorylase activity. The positive chronotropic responses were unaltered by acetylcholine infusion. 4. The ratio of the dose required for a standard heart rate response to the dose producing a standard contractile force response was different for each agonist. 5. The selective antagonism of the contractile response to isoprenaline, salbutamol and aminophylline suggest that different mechanisms are involved in the initiation of positive inotropic and chronotropic responses.

Acetylcholine↗
PubMed1980

Effects of hydralazine and verapamil on phosphorylase activity and guanosine cyclic 3',5'-monophosphate levels in guinea-pig taenia coli.

1 The roles of guanosine cyclic 3',5'-monophosphate (cyclic GMP) and calcium in the relaxation produced by hydralazine and verapamil in potassium-depolarized guinea-pig taenia coli have been investigated.2 Depolarization of isolated strips of guinea-pig taenia coli by 124 mM KCl caused sustained contractures and increases in tissue levels of cyclic GMP.3 The KCl-induced increases in cyclic GMP levels appeared to be calcium dependent. No increases in cyclic GMP levels were seen in strips of taenia coli depolarized in the absence of calcium. Readdition of calcium to the depolarizing solution contracted the muscles and increased cyclic GMP levels. When calcium was removed from the depolarizing solution during the sustained, tonic phase of a KCl-induced contracture, both tension and cyclic GMP levels returned to control values.4 Administration of 50 muM verapamil to KCl-contracted muscles completely relaxed the muscles and caused cyclic GMP levels to return to control values within 14 min. Hydralazine, 2 mM, on the other hand, relaxed the depolarized muscles without lowering cyclic GMP levels. No significant changes in cyclic AMP levels were seen in any of these experiments.5 Similar results were obtained in an analogous series of experiments in which glycogen phosphorylase activity was measured instead of cyclic GMP levels. Activation of phosphorylase during contractions of guinea-pig taenia coli had previously been reported to be a calcium-dependent phenomenon.6 It was concluded that increases in tissue levels of cyclic GMP (or of cyclic AMP) were not responsible for the relaxant effects of hydralazine or verapamil in these experiments. It was also suggested, based on our results, that verapamil exerted its relaxant effects in the depolarized taenia coli by lowering cytoplasmic calcium levels, whereas hydralazine relaxed the depolarized muscles without lowering intracellular calcium activity.

Animals↗
PubMedDate not supplied

Breakdown of glucopolysaccharides in Entamoeba histolytica by phosphorylase.

Homogenates of trophozoites of Entamoeba histolytica released glucose 1-phosphate from amylopectin, glycogen, and amylose in a ratio of 100:78:74 at glucopolysaccharide concentrations of 0.1%. By use of self-generating Percoll gradients this activity was shown to be particulate and associated with glycogen. The phosphorylase was extracted from the 40,000 g pellet in aqueous medium and purified to homogeneity by gel filtration on Fractogel TSK HW-55(F) followed by chromatography on Blue Sepharose CL-6B. The purified enzyme was active not only against the glucopolysaccharides but also on dextrins with more than 3 glucose moieties, which were primarily formed by the action of amoebic amylases. At substrate concentrations of 1 mM nonreducing ends of each glucan, the phosphorolysis rate of the branched polysaccharides was about 1.75 x 10(4) times higher than those of the maltodextrins. By means of HPLC the sequential degradation of 4-nitrophenyl-maltoheptaoside (G(7)-pNP) was studied. Native phosphorylase exhibited a relative molecular mass of M(r) = 200,000 by gel filtration and gel electrophoresis. The SDS electrophoresis, under reducing conditions, indicated that the native enzyme was a dimer. Optimal degradation of the polysaccharides and dextrins was achieved at pH values of 7.5 and 7.0 respectively.

Animals↗
PubMed1988

Skeletal muscle glucolysis, glycogenolysis and glycogen phosphorylase during electrical stimulation in man.

Phosphorylase activity, glycogenolytic and glucolytic rates were estimated in human quadriceps muscle during electrical stimulation at 20 Hz. Two stimulation periods of 10 s duration were separated by a pause of 60 s. The blood circulation to the leg was intact or occluded during the experiment. ATP turnover rates and force production were of the same order during the two contraction periods both with and without intact blood flow. Also the increase in phosphorylase a activity (from approximately 30% to approximately 65%) was the same during the contraction periods. Glycogenolytic and glucolytic rates were however about 30% higher (P less than 0.05) during the second contraction compared with the first when circulation was occluded, but similar when the circulation was intact. During the 60 s rest period, the phosphocreatine (PCr) was maintained at a low level and inorganic phosphate (Pi) remained increased under occluded circulation while PCr was resynthesized in the rest period with intact circulation. We conclude that the increased glycogenolytic rate observed during the second contraction with occluded blood circulation was due to the high [Pi] in the muscle and that the increased glucolytic rate was caused by high [Pi] and low [PCr]. In the rest period with anoxia the glycogenolysis was completely inhibited and glucolysis was inhibited by 95% in spite of the changes in [PCr] and [Pi].

Adult↗
PubMed1975

Carcinofetal alterations in glycogen phosphorylase isozymes in rat hepatomas.

As part of a continuing study on the loss or retention of enzymes involved in specific hepatic functions in a series of rat liver neoplasms ranging widely in growth rate and degree of differentiation, isozyme patterns of glycogen phosphorylase in rat hepatomas were compared with those in rat tissues during development. A third phosphorylase isozyme, distinguishable kinetically, immunologically, electrophoretically, and by isoelectric focusing from liver and muscle types is commonly present in various rat hepatomas including Novikoff, Morris, and Yoshida hepatomas with various degrees of retention of liver type according to their degree of differentiation. This isozyme is also the sole type in the placenta and early embryo, and in liver and muscle is replaced with the organ specific liver and muscle types during development. In other organs, the replacement is only partial, and, especially in adult rat brain, this type is retained. Thus, this isozyme seems to be a prototype whose appearance in hepatomas is one of many examples of carcinofetal alterations in isozymes.

Animals↗
PubMed2004

Regulation of rat hepatocyte function by P2Y receptors: focus on control of glycogen phosphorylase and cyclic AMP by 2-methylthioadenosine 5'-diphosphate.

Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5'-diphosphate (2-MeSADP), an agonist at P2Y(1), P2Y(12), and P2Y(13) receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N(6)-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y(1) receptors. In addition, in these cells, both 2-MeSADP and UTP inhibited glucagon-stimulated cyclic AMP accumulation. This inhibitory effect of 2-MeSADP was not reversed by the P2Y(1) antagonists, adenosine-3'-phosphate-5'-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 microM, indicating that it was not mediated by P2Y(1) receptors. This contrasts with the increase in cytosolic free Ca(2+) concentration ([Ca(2+)](c)) induced by 2-MeSADP, which has shown to be inhibited by A3P5P. Pertussis toxin abolished the inhibitory effect of both UTP and 2-MeSADP. After culture of cells for 48 h, the ability of 2-MeSADP to inhibit cyclic AMP accumulation was greatly diminished. Reverse transcriptase-polymerase chain reaction analysis revealed that during this culture period, there was a decline in the ability to detect transcripts for P2Y(12) and P2Y(13) receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y(12) and P2Y(13) receptor antagonist, 2-propylthio-beta,gamma-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 microM) did not alter the ability of 2-MeSADP to inhibit glucagon-stimulated cyclic AMP accumulation. We conclude that 2-MeSADP regulates rat hepatocyte glycogen phosphorylase by acting on P2Y(1) receptors coupled to raised [Ca(2+)](c), and by inhibiting cyclic AMP levels by an unknown G(i)-coupled receptor subtype, distinct from P2Y(1), P2Y(12), or P2Y(13) receptors.

Adenosine Diphosphate↗
PubMed2005

Regulation of human hepatocytes by P2Y receptors: control of glycogen phosphorylase, Ca2+, and mitogen-activated protein kinases.

In the rat both short-term liver function, such as glycogen metabolism, and long-term events such as proliferation after partial hepatectomy, are in part controlled by release of nucleotides such as ATP acting on hepatocyte P2Y(1) and P2Y(2) receptors (members of a family of P2Y receptors for extracellular nucleotides such as ATP and UTP). Here, we have studied P2Y receptor regulation of signaling pathways involved in glycogen phosphorylase activation and proliferation of primary human hepatocytes. Stimulation of cultured hepatocytes with either ATP and UTP, but not UDP or 2-methylthio ADP, led to concentration-dependent increases in cytosolic free Ca(2+) concentration ([Ca(2+)](c); EC(50) for ATP = 3.3 microM, for UTP = 2.3 microM) and [(3)H]inositol (poly)phosphates (EC(50) for ATP = 9.4 microM, for UTP = 15.4 microM). ATP and UTP also stimulated glycogen phosphorylase in human hepatocytes, each with a threshold for activation of less than 1 microM. Application of 2-methylthio ADP up to 100 microM was ineffective. Phosphorylation of both extracellular signal-related kinase and c-Jun N-terminal kinase was stimulated by ATP and UTP, but not by 2-methylthio ADP or UDP, either alone or when costimulated with epidermal growth factor. In conclusion, in human hepatocytes P2Y receptors control both glycogen metabolism and proliferation-associated responses such as increased [Ca(2+)](c) and mitogen-activated protein kinase cascades. Regulation seems to be primarily through P2Y(2) receptors. In contrast with previous studies on rat hepatocytes, there is an absence of responses mediated by P2Y(1) receptors.

Adenosine Triphosphate↗
PubMed1991

Structural basis for the activation of glycogen phosphorylase b by adenosine monophosphate.

The three-dimensional structure of the activated state of glycogen phosphorylase (GP) as induced by adenosine monophosphate (AMP) has been determined from crystals of pyridoxalpyrophosphoryl-GP. The same quaternary changes relative to the inactive conformation as those induced by phosphorylation are induced by AMP, although the two regulatory signals function through different local structural mechanisms. Moreover, previous descriptions of the phosphorylase active state have been extended by demonstrating that, on activation, the amino- and carboxyl-terminal domains of GP rotate apart by 5 degrees, thereby increasing access of substrates to the catalytic site. The structure also reveals previously unobserved interactions with the nucleotide that accounts for the specificity of the nucleotide binding site for AMP in preference to inosine monophosphate.

Adenosine Monophosphate↗
PubMed1977

Purine nucleoside phosphorylase deficiency: altered kinetic properties of a mutant enzyme.

Erythrocyte purine nucleoside phosphorylase from two brothers had 0.5% of normal activity. It differed from the normal enzyme by a tenfold increase in the Michaelis constant for inosine, an inability of inosine to protect against thermal lability, and a more positive net charge. The altered kinetic properties may account for the milder disease in the patients compared to the previously described cases. The data provide evidence for a structural gene mutation and genetic heterogeneity in the new disease of purine nucleoside phosphorylase deficiency and T cell dysfunction.

Child↗
PubMed1990

Purification and characterization of purine nucleoside phosphorylase from Proteus vulgaris.

Purine nucleoside phosphorylase was isolated and purified from cell extracts of Proteus vulgaris recovered from spoiling cod fish (Gadus morhua). The molecular weight and isoelectric point of the enzyme were 120,000 +/- 2,000 and pH 6.8. The Michaelis constant for inosine as substrate was 3.9 x 10(-5). Guanosine also served as a substrate (Km = 2.9 x 10(-5). However, the enzyme was incapable of phosphorylizing adenosine. Adenosine proved to be useful as a competitive inhibitor and was used as a ligand for affinity chromatography of purine nucleoside phosphorylase following initial purification steps of gel filtration and ion-exchange chromatography.

Inosine↗
PubMed1982

Pyrimidine nucleoside phosphorylase activity in tumour and matched normal gastrointestinal mucosa.

5-Fluorouracil (5-FU) requires activation to the metabolites FdUMP and FUTP for its cytotoxic effect. This activation involves the intracellular enzymes uridine and thymidine phosphorylase. We have assayed the levels of these enzymes in colorectal and gastric cancers and have shown that, in the majority of cases, the enzyme levels were higher than in the adjacent normal mucosa. It is suggested that, while the ratio tumour/normal mucosa enzyme activity may give some indication of the relative toxicity of 5-FU in these tissues, the absolute activities of the phosphorylases in tumour tissue could give a better indication of tumour responsiveness.

Adult↗