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At least 667 records · Page 37Linked to original sources
PubMed1984

Hormonal regulation of L-type pyruvate kinase in hepatocytes from phosphorylase kinase-deficient (gsd/gsd) rats.

The hormonal regulation of L-type pyruvate kinase in hepatocytes from phosphorylase b kinase-deficient (gsd/gsd) rats was investigated. Adrenaline (10 microM) and glucagon (10 nM) each led to an inactivation and phosphorylation of pyruvate kinase. Dose-response curves for adrenaline-mediated inactivation of pyruvate kinase, phosphorylation of pyruvate kinase and the stimulation of gluconeogenesis from 1.8 mM-lactate were similar for hepatocytes from control and gsd/gsd rats. Time-course studies indicated that adrenaline-mediated inactivation and phosphorylation of pyruvate kinase proceeded more slowly in phosphorylase kinase-deficient hepatocytes than in control hepatocytes. The age-dependent change in the adrenergic control of pyruvate kinase was similar between control and phosphorylase kinase-deficient hepatocytes. Adrenaline, glucagon and noradrenaline activated the cyclic AMP-dependent protein kinase and inhibited pyruvate kinase in phosphorylase kinase-deficient hepatocytes. Vasopressin (0.2-2 nM), angiotensin (10nM) and A23187 (10 microM) had no effect on the activity ratio of the cyclic AMP-dependent protein kinase or pyruvate kinase in these cells. It is concluded that phosphorylase kinase plays no significant role in the hormonal control of pyruvate kinase and that phosphorylation and inactivation of this enzyme results predominantly from the action of the cyclic AMP-dependent protein kinase.

Animals↗
PubMed1991

The expression of glycogen phosphorylase in normal and dystrophic muscle.

Specific cofactor labelling was employed to determine the degradation rate of glycogen phosphorylase in normal adult C57BL/6J mice and their dystrophic counterparts (C57BL/6Jdy/dy). The rate constant for the decay of phosphorylase-bound label was 0.125 day-1 in normal muscle and 0.49 day-1 in dystrophic muscle, i.e. a lower rate of catabolism of phosphorylase in dystrophic muscle. Quantitative Northern-blot analyses of total RNA isolated from normal and dystrophic muscle indicated that the abundance of phosphorylase mRNA as a percentage of total RNA was approx. 40% lower in dystrophic muscle. The specific activity of phosphorylase in dystrophic muscle is approx. 60% lower than in normal muscle, and is elicited by a lower rate of turnover of the enzyme, i.e. both synthesis and degradation are decreased.

Animals↗
PubMed1992

Immunological detection of degradation intermediates of skeletal-muscle glycogen phosphorylase in vitro and in vivo.

Over 95% of the pyridoxal phosphate (PLP) in skeletal is bound to one protein, glycogen phosphorylase. This, and the fact that phosphorylase constitutes approx. 5% of the soluble protein in skeletal muscle, introduce the possibility that PLP might be used as a specific label to identify degradation intermediates of the enzyme. In this investigation, we have developed immunological methods, using a monoclonal antibody to PLP and polyclonal antibodies to phosphorylase, to detect degradation intermediates in vitro and in vivo. We have identified a family of degradation intermediates of glycogen phosphorylase in the high-speed-supernatant fraction of mouse skeletal muscle. These peptides react with both types of antibodies and are in the size and concentration range expected for degradation intermediates in a model in which the committed step is followed by rapid clearance of the products. Changes in amounts of degradation intermediates are examined in physiological or pathological conditions in which the rate of degradation of phosphorylase is altered.

Animals↗
PubMed1997

Neural regulation of the formation of skeletal muscle phosphorylase kinase holoenzyme in adult and developing rat muscle.

Neural influences on the co-ordination of expression of the multiple subunits of skeletal muscle phosphorylase kinase and their assembly to form the holoenzyme complex, alpha4beta4gamma4delta4, have been examined during denervation and re-innervation of adult skeletal muscle and during neonatal muscle development. Denervation of the tibialis anterior and extensor digitorum longus muscles of the rat hindlimb was associated with a rapid decline in the mRNA for the gamma subunit, and an abrupt decrease in gamma-subunit protein. The levels of the alpha- and beta-subunit proteins in the denervated muscles also declined rapidly, their time course of reduction being similar to that for the gamma-subunit protein, but they did not decrease to the same extent. In contrast with the rapid decline in gamma-subunit mRNA upon denervation, alpha- and beta-subunit mRNAs stayed at control innervated levels for approx. 8-10 days, but then decreased rapidly. Their decline coincided very closely with the onset of re-innervation. Re-innervation of the denervated muscles, which occurs rapidly and uniformly after the sciatic nerve crush injury, produced an eventual slow and prolonged recovery of the mRNA for all three subunits and parallel increases in each of the subunit proteins. A similar co-ordinated increase of both subunit mRNA and subunit proteins of the phosphorylase kinase holoenzyme was observed during neonatal muscle development, during the period when the muscles were attaining their adult pattern of motor activity. The phosphorylase kinase holoenzyme remains in a non-activated form during all of these physiological changes, as is compatible with the presence of the full complement of the regulatory subunits. These data are consistent with a model whereby the transcriptional and translational expression of phosphorylase kinase gamma subunit occurs only with concomitant expression of the alpha and beta subunits. This would ensure that free and unregulated, activated gamma subunit alone, which would give rise to unregulated glycogenolysis, is not produced. The data also suggest that control of phosphorylase kinase subunit expression and the formation of the holoenzyme in skeletal muscle is provided by the motor nerve, probably through imposed levels or patterns of muscle activity.

Aging↗
PubMed1998

Homodimers and heterodimers of Pho1-type phosphorylase isoforms in Solanum tuberosum L. as revealed by sequence-specific antibodies.

Higher plants possess two types of glucan phosphorylase (EC 2.4.1.1). One isozyme type, designated as Pho1, is located in the plastid whereas the other type, Pho2, is restricted to the cytosol. For Solanum tuberosum L. two Pho1 type phosphorylases have been sequenced [Nakano, K. & Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236; Sonnewald, U., Basner, A., Greve, B. & Steup, M. (1995) Plant Mol. Biol. 27, 567-576]. Both proteins (referred to as Pho1a and Pho1b, respectively) are highly similar (81-84% amino acid identity over most parts of the two sequences) with the exception of the N-terminal transit peptide and the large insertion located between the N- and the C-terminal domains. In this communication antibodies that bind specifically to either Pho1a or Pho1b were used to study both isoforms at the protein level. The antibodies were applied to both potato tuber and leaf extracts following either denaturing or non-denaturing electrophoresis. Pho1a but not Pho1b was immunochemically detectable in tuber extracts whereas leaf extracts contained both the Pho1a and Pho1b protein. During denaturing electrophoresis the two antigens comigrated. When the leaf Pho1 isoforms were separated by affinity electrophoresis three bands of activity were resolved; all of them were recognized by the anti-Pho1a antibodies, but only two of these reacted with the anti-Pho1b antibodies. The isoform binding exclusively to the anti-Pho1a antibodies comigrated with the Pho1 isozyme from potato tubers. Immunoprecipitation experiments performed with anti-Pho1a antibodies removed the entire Pho1 phosphorylase activity from both tuber and leaf extracts. Addition of anti-Pho1b antibodies to tuber extracts did not affect the enzyme pattern, whereas in leaf extracts one isoform remained unchanged but the two other bands were strongly retarded. This indicates that the Pho1a protein is present in all three forms and Pho1b is associated with Pho1a. Association of Pho1a and Pho1b was further demonstrated by cross-linking experiments using bis(sulfosuccinimidyl)suberate as linker. Immunoprecipitation experiments were also performed using extracts of transformed Escherichia coli cells that expressed either Pho1a or Pho1b or both simultaneously. Under these conditions a homodimeric Pho1b phosphorylase was observed that had a lower electrophoretic mobility than the heterodimer from leaves. In leaves of transgenic potato plants antisense inhibition of the Pho1a gene affected the formation of (Pho1a)2 more strongly than that of the heterodimer. Thus, in leaves, Pho1a exists both as a homodimer, (Pho1a)2 and as heterodimer, (Pho1a-Pho1b); a part of it appears to be covalently modified. Pho1b, in the homodimeric form, is often below the limit of detection. In tubers the homodimer, (Pho1a)2, is the only detectable Pho1-type enzyme. To our knowledge this is the first report on a heterodimeric structure of plant phosphorylase.

Amino Acid Sequence↗
PubMed2001

Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N-oxide on the self-association of muscle glycogen phosphorylase b.

The suitability of sedimentation equilibrium for characterizing the self-association of muscle glycogen phosphorylase b has been reappraised. Whereas sedimentation equilibrium distributions for phosphorylase b in 40 mM Hepes buffer (pH 6.8) supplemented with 1 mM AMP signify a lack of chemical equilibrium attainment, those in buffer supplemented additionally with potassium sulfate conform with the requirements of a dimerizing system in chemical as well as sedimentation equilibrium. Because the rate of attainment of chemical equilibrium under the former conditions is sufficiently slow to allow resolution of the dimeric and tetrameric enzyme species by sedimentation velocity, this procedure has been used to examine the effects of thermodynamic nonideality arising from molecular crowding by trimethylamine N-oxide on the self-association behaviour of phosphorylase b. In those terms the marginally enhanced extent of phosphorylase b self-association observed in the presence of high concentrations of the cosolute is taken to imply that the effects of thermodynamic nonideality on the dimer-tetramer equilibrium are being countered by those displacing the T<==>R isomerization equilibrium for dimer towards the smaller, nonassociating T state. Because the R state is the enzymically active form, an inhibitory effect is the predicted consequence of molecular crowding by high concentrations of unrelated solutes. Thermodynamic nonideality thus provides an alternative explanation for the inhibitory effects of high concentrations of glycerol, sucrose and ethylene glycol on phosphorylase b activity, phenomena that have been attributed to extremely weak interaction of these cryoprotectants with the T state of the enzyme.

Animals↗
PubMed1986

Sequence analysis of the cDNA encoding human liver glycogen phosphorylase reveals tissue-specific codon usage.

We have cloned the cDNA encoding glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyl-transferase, EC 2.4.1.1) from human liver. Blot-hybridization analysis using a large fragment of the cDNA to probe mRNA from rabbit brain, muscle, and liver tissues shows preferential hybridization to liver RNA. Determination of the entire nucleotide sequence of the liver message has allowed a comparison with the previously determined rabbit muscle phosphorylase sequence. Despite an amino acid identity of 80%, the two cDNAs exhibit a remarkable divergence in G+C content. In the muscle phosphorylase sequence, 86% of the nucleotides at the third codon position are either deoxyguanosine or deoxycytidine residues, while in the liver homolog the figure is only 60%, resulting in a strikingly different pattern of codon usage throughout most of the sequence. The liver phosphorylase cDNA appears to represent an evolutionary mosaic; the segment encoding the N-terminal 80 amino acids contains greater than 90% G+C at the third codon position. A survey of other published mammalian cDNA sequences reveals that the data for liver and muscle phosphorylases reflects a bias in codon usage patterns in liver and muscle coding sequences in general.

Animals↗
PubMed1979

Association of glycogen synthase phosphatase and phosphorylase phosphatase activities with membranes of hepatic smooth endoplasmic reticulum.

A detailed investigation was conducted to determine the precise subcellular localization of the rate-limiting enzymes of hepatic glycogen metabolism (glycogen synthase and phosphorylase) and their regulatory enzymes (synthase phosphatase and phosphorylase phosphatase). Rat liver was homogenized and fractionated to produce soluble, rough and smooth microsomal fractions. Enzyme assays of the fractions were performed, and the results showed that glycogen synthase and phosphorylase were located in the soluble fraction of the livers. Synthase phosphatase and phosphorylase phosphatase activities were also present in soluble fractions, but were clearly identified in both rough and smooth microsomal fractions. It is suggested that the location of smooth endoplasmic reticulum (SER) within the cytosome forms a microenvironment within hepatocytes that establishes conditions necessary for glycogen synthesis (and degradation). Thus the location of SER in the cell determines regions of the hepatocyte that are rich in glycogen particles. Furthermore, the demonstration of the association of synthase phosphatase and phosphorylase phosphatase with membranes of SER may account for the close morphological association of SER with glycogen particles (i.e., disposition of SER membranes brings the membrane-bound regulatory enzymes in close contact with their substrates).

Animals↗
PubMed1998

Mutations in the liver glycogen phosphorylase gene (PYGL) underlying glycogenosis type VI.

Deficiency of glycogen phosphorylase in the liver gives rise to glycogen-storage disease type VI (Hers disease; MIM 232700). We report the identification of the first mutations in PYGL, the gene encoding the liver isoform of glycogen phosphorylase, in three patients with Hers disease. These are two splice-site mutations and two missense mutations. A mutation of the 5' splice-site consensus of intron 14 causes the retention of intron 14 and the utilization of two illegitimate 5' splice sites, whereas a mutation of the 3' splice-site consensus of intron 4 causes the skipping of exon 5. Two missense mutations, N338S and N376K, both cause nonconservative replacements of amino acids that are absolutely conserved even in yeast and bacterial phosphorylases. We also report corrections of the PYGL coding sequence, sequence polymorphisms, and a partial PYGL gene structure with introns in the same positions as in PYGM, the gene of the muscle isoform of phosphorylase. Our findings demonstrate that PYGL mutations cause Hers disease, and they may improve laboratory diagnosis of deficiencies of the liver phosphorylase system.

Amino Acid Sequence↗
PubMed2005

Fatal congenital heart glycogenosis caused by a recurrent activating R531Q mutation in the gamma 2-subunit of AMP-activated protein kinase (PRKAG2), not by phosphorylase kinase deficiency.

Fatal congenital nonlysosomal cardiac glycogenosis has been attributed to a subtype of phosphorylase kinase deficiency, but the underlying genes and mutations have not been identified. Analyzing four sporadic, unrelated patients, we found no mutations either in the eight genes encoding phosphorylase kinase subunits or in the two genes encoding the muscle and brain isoforms of glycogen phosphorylase. However, in three of five patients, we identified identical heterozygous R531Q missense mutations of the PRKAG2 gene, which encodes the gamma 2-subunit of AMP-activated protein kinase, a key regulator of energy balance. Biochemical characterization of the recombinant R531Q mutant protein showed >100-fold reduction of binding affinities for the regulatory nucleotides AMP and ATP but an enhanced basal activity and increased phosphorylation of the alpha -subunit. Other PRKAG2 missense mutations were previously identified in patients with autosomal dominant hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome, characterized by juvenile-to-adult clinical onset, moderate cardiac glycogenosis, disturbed excitation conduction, risk of sudden cardiac death in midlife, and molecular perturbations that are similar to--but less severe than--those observed for the R531Q mutation. Thus, recurrent heterozygous R531Q missense mutations in PRKAG2 give rise to a massive nonlysosomal cardiac glycogenosis of fetal symptomatic onset and rapidly fatal course, constituting a genotypically and clinically distinct variant of hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome. R531Q and other PRKAG2 mutations enhance the basal activity and alpha -subunit phosphorylation of AMP-activated protein kinase, explaining the dominant nature of PRKAG2 disease mutations. Since not all cases displayed PRKAG2 mutations, fatal congenital nonlysosomal cardiac glycogenosis seems to be genetically heterogeneous. However, the existence of a heart-specific primary phosphorylase kinase deficiency is questionable, because no phosphorylase kinase mutations were found.

AMP-Activated Protein Kinases↗
PubMed1989

Exercise training and dietary chromium effects on glycogen, glycogen synthase, phosphorylase and total protein in rats.

The effects of exercise training and dietary chromium intake on rat liver and muscle glycogen metabolism, tissue and body weight and feed consumption were examined. After 16 wk of training, liver, gastrocnemius and biceps femoris glycogen concentrations were higher in the trained compared to sedentary groups, independent of dietary chromium. There was a chromium x training interaction on glycogen synthase activities in the liver and gastrocnemius muscle. Liver glycogen phosphorylase activities (expressed per g liver) were lower in the chromium-supplemented rats as compared to the non-supplemented rats after 5 wk of dietary treatment, but were similar after 8 wk and higher after 18 wk. Gastrocnemius phosphorylase activity (expressed per mg protein) was lower in the trained rats as compared to the sedentary rats after 16 wk, independent of dietary chromium. Biceps femoris phosphorylase activities were not altered due to training or dietary chromium. Total protein concentration increased in the liver but decreased in the gastrocnemius due to dietary chromium. In summary, liver glycogen synthase and phosphorylase activities were dependent upon dietary chromium. Dietary chromium altered gastrocnemius synthase, but not phosphorylase activities. Changes in enzyme activities may be related to the chromium-dependent effects on liver protein and the chromium and training-dependent effects on gastrocnemius total protein.

Animals↗
PubMed1981

Phosphoprotein phosphatase associated with rat liver plasma membrane. Properties of phosphorylase phosphatase and phosphohistone phosphatase.

Plasma membrane isolated from rat liver contained activities of phosphoprotein phosphatase dephosphorylating [32P]phosphorylase a or [32P]phosphohistone. The properties of the membrane-bound phosphatase were examined using these exogenous substrates. The optimal reaction rate was at pH near neutrality. At concentrations as low as 0.1-1.0 mM, Mg2+ or Mn2+ slightly stimulated the activity for phosphorylase a or phosphohistone, respectively; at higher concentrations, they were inhibitory with both substrates. Co2+ was inhibitory with both substrates, while Ca2+ had no significant effect. The phosphatase activities were inhibited by ATP, ADP, or AMP; the extents of inhibition were in opposite order with the two substrates. Phosphorylase phosphatase activity was strongly inhibited by KF or Pi. Phosphorylase phosphatase activity could be completely solubilized by incubating the membrane with 0.5 M NaCl or trypsin, and this was associated with several-fold activation. While Vmax values were increased, Km values for phosphorylase a were not much affected by these treatments. Unlike the soluble phosphatase, freezing in the presence of mercaptoethanol or by precipitation with ethanol failed to activate or to solubilize the membrane-bound phosphatase. The molecular weights of the NaCl-and the trypsin-solubilized phosphatase were estimated on gel filtration to be about 42,000 and 32,000, respectively. The present results indicate that the phosphoprotein phosphatase associated with liver plasma membrane shares several properties in common with phosphatases from other sources reported, and that, like those in the soluble fraction, it may be bound to some inhibitory proteins.

Animals↗
PubMed1989

Maturation and subcellular compartmentation of potato starch phosphorylase.

The subcellular localization and maturation of starch phosphorylase (EC 2.4.1.1) was studied in developing potato tubers. The enzyme is localized inside the stroma of amyloplasts in young tubers, whereas in mature tubers it is found within the cytoplasm in the immediate vicinity of the plastids. A phosphorylase cDNA clone was isolated and used in RNA gel blot experiments to demonstrate that phosphorylase mRNAs are of the same size and abundance in both young and mature tubers. In vitro translation of mRNAs followed by immunoprecipitation with a phosphorylase antiserum indicates that the enzyme is synthesized as a higher molecular weight precursor in both young and mature tubers. The presence of a transit peptide at the N terminus of the protein was confirmed by the sequencing of the phosphorylase cDNA clone. The transit peptide has several structural features common to transit peptides of chloroplast proteins but contains a surprisingly large number of histidine residues. The mature form of the enzyme is present in both young and mature tubers, suggesting that a similar processing of the transit peptide may take place in two different subcellular locations.

Amino Acid Sequence↗
PubMed1985

ADP-ribosylation of phosphorylase kinase and block of phosphate incorporation into the enzyme.

Phosphorylase kinase purified from rabbit skeletal muscle was ADP-ribosylated by hen liver nuclear ADP-ribosyltransferase. This modification, as was seen in cAMP-dependent phosphorylation, was observed only in alpha and beta subunits of the phosphorylase kinase and the latter was more rapidly modified. Analysis of the ADP-ribosylated amino acid residue sequenced in alpha and beta subunits showed that both subunits were modified at the area of the arginine residue. The Km for NAD was 0.10 mM and the pH optimum was 9.0. When the ADP-ribosylated phosphorylase kinase was phosphorylated by cAMP-dependent protein kinase, a reduction in phosphate incorporation occurred with increase in the ADP-ribosylation. ADP-ribosylation also suppressed autophosphorylation, to a lesser degree than observed with cAMP-dependent phosphorylation. The ADP-ribosylation-dependent reduction of phosphorylation resulted in a suppression of the phosphorylation-dependent activation of the phosphorylase kinase. These results together with findings of ADP-ribosyltransferase activity in the rabbit skeletal muscle [Soman, G. et al. (1984) Biochem. Biophys. Res. Commun. 120, 973-980] suggest that ADP-ribosylation participates in the regulation of the phosphorylase kinase activity through changes in the rate of phosphorylation.

Adenosine Diphosphate Ribose↗
PubMed1988

The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers.

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35 degrees C without affecting cyclic AMP concentration.

Cyclic AMP↗
PubMed1988

The control of glycogen metabolism in yeast. 2. A kinetic study of the two forms of glycogen synthase and of glycogen phosphorylase and an investigation of their interconversion in a cell-free extract.

Two interconvertible forms of glycogen synthase and glycogen phosphorylase, one active (a) or the other less active (b), were predominantly present in a thermosensitive adenylate-cyclase-deficient mutant that had been preincubated at the restrictive temperature of 35 degrees C, either in the presence or in the absence of glucose. Glycogen phosphorylase was at least 20-fold less active after incubation of the cells in the presence of glucose, but this residual activity had kinetic properties identical to those of the active form of enzyme, obtained after incubation in the absence of glucose; this suggests that the b form might be completely inactive and that the low activity measured after glucose treatment must be attributed to a residual amount of phosphorylase a. By contrast, the kinetic properties of the two forms of glycogen synthase were very different. When measured in the absence of glucose 6-phosphate, the two forms of enzyme had a similar affinity for UDP-Glc but differed essentially by their Vmax. Glucose 6-phosphate had no effect on synthase a, but increased both Vmax and Km of synthase b; these effects, however, were in great part counteracted by sulfate and by inorganic phosphate, the latter also having the property of increasing the Km of the a form, without affecting Vmax. It was estimated that at physiological concentrations of substrates and effectors, synthase a was about 20-fold more active than synthase b. When an extract of cells that had been preincubated in the absence of glucose was gel-filtered and then incubated at 30 degrees C, phosphorylase was progressively fully inactivated and synthase was partially activated; these reactions were severalfold faster and, in the case of glycogen synthase, more complete in the presence of 10 mM glucose 6-phosphate. When a gel-filtered extract of cells that had been preincubated in the presence of glucose was incubated at 30 degrees C in the presence of ATP-Mg and EGTA, phosphorylase became activated and synthase was inactivated; the first of these two reactions was severalfold stimulated by micromolar concentrations of Ca2+, whereas both reactions were completely inhibited by 10 mM glucose 6-phosphate and only slightly and irregularly stimulated by cyclic AMP.

Adenosine Triphosphate↗
PubMed1992

Tissue distribution and change in potato starch phosphorylase mRNA levels in wounded tissue and sprouting tubers.

Starch phosphorylase has been cloned from a lambda gt10 cDNA library of potato tuber mRNA. Selected recombinants have been used to demonstrate that phosphorylase mRNA is most abundant in tubers but is also detectable in stolon, root, stem and leaf tissue. The level of phosphorylase mRNA was greatly reduced in wounded stem and tuber tissue. The wounding-induced decrease in phosphorylase mRNA levels is not reversed in the presence of sucrose or mannitol. Regional differences are described in the levels of phosphorylase and patatin mRNA in different parts of the tuber and in the shoot of sprouting potatoes.

Base Sequence↗
PubMed1996

Extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus. Gene cloning and amino acid sequence determination.

A gene encoding an extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase was cloned from the archaeon Sulfolobus solfataricus. Two degenerate oligodeoxyribonucleotide probes synthesized on the basis of the N-terminal amino acid sequence of the protein were used to screen a genomic library of S. solfataricus cloned into the pGEM7Zf(+) vector. The DNA fragment of 2118 bp containing the 5'-methylthioadenosine phosphorylase gene was sequenced. The open reading frame comprises 711 nucleotides, which includes the stop codon, and encodes a protein of 236 residues whose molecular mass is in good agreement with the value determined by gel filtration for the purified enzyme. The N- and C-terminal sequences of the protein and the sequences of the peptides prepared by cyanogen bromide cleavage exactly match with the corresponding sequences deduced from the gene, thus confirming the identity of the 5'-methylthioadenosine phosphorylase gene. Typical archaebacterial regulatory sites were identified in the flanking regions and a potential Shine-Dalgarno-like sequence was recognized around the ATG initiation codon. The deduced amino acid sequence showed 32% identity and 30% identity with Escherichia coli purine-nucleoside phosphorylase and with E, coli uridine phosphorylase, respectively. Evolutionary and structural implications of this similarity are discussed.

Amino Acid Sequence↗