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PubMed1993

[Action of dopamine on glucose level and glycogen phosphorylase activity of blood plasma in experimental coronary occlusion].

The effects of dopamine (5 micrograms/kg.min) on the blood plasma glucose level and glycogen-phosphorylase activity in experimental coronary occlusion were studied. The absence of the raising of the plasma glucose level and glycogen-phosphorylase "a" and "b" activities has been observed in 25 min after the coronary occlusion. It is supposed that the functional improvement of the ischemized myocardium by dopamine (5 micrograms/kg.min) is the result of the stimulation of presynaptic D-2-dopaminergic and postsynaptic D-1-dopaminergic receptors in coronary arteries.

Animals↗
PubMed1994

[Mechanism for regulating the activity of muscle glycogen phosphorylase b by adenosine-5'-monophosphate].

The kinetics of glycogen chains growth catalyzed by glycogen phosphorylase b from rabbit skeletal muscles at saturating glycogen concentrations have been studied. The applicability of twelve different variants of the Monod-Wyman-Changeux model and six kinetic schemes assuming the interaction of AMP- and glucose 1-phosphate binding sites in the dimeric enzyme molecule is discussed. The kinetic model of the reaction is proposed which suggests: (1) independent binding of AMP and a molecule of glucose 1-phosphate to the dimeric enzyme molecule saturated by glycogen; (2) exclusive binding of the second glucose 1-phosphate molecule by the dimeric enzyme molecule containing two molecules of bound AMP and one molecule of bound glucose 1-phosphate; (3) exclusive ability of the enzyme complex with glycogen, two molecules of AMP and two molecules of glucose 1-phosphate in catalytic transformation. The parameters of the rate equation were calculated by nonlinear regression analysis. The proposed kinetic scheme satisfies the following criteria: (1) convergence in the regressional analysis; (2) reliability of the parameter values; (3) the minimal sum of the weighed squares of residuals. This model was found to fit adequately the kinetics of muscle phosphorylase b reaction at saturating glycogen concentrations.

Adenosine Monophosphate↗
PubMed1994

Slow conformational transitions of muscle glycogen phosphorylase b induced by specific ligands.

The effect of specific ligands (the substrate glucose 1-phosphate, AMP, and flavins) on the initial rate of tryptic digestion of muscle glycogen phosphorylase b has been studied (0.02 M HEPES, pH 6.8; 37 degrees C). Differences were observed between the kinetic curves of the enzyme trypsinolysis initiated by the addition of a trypsin/ligand mixture and once trypsin was added to the enzyme preincubated with a ligand for 10 min. It was suggested that the specific ligands studied induce relatively slow conformational changes in the phosphorylase b molecule.

Adenosine Monophosphate↗
PubMed1993

The multiphosphorylation domain of the phosphorylase kinase alpha M and alpha L subunits is a hotspot of differential mRNA processing and of molecular evolution.

We have cloned and sequenced human cDNAs encoding the complete phosphorylase kinase alpha subunit muscle isoform (alpha M). The predicted polypeptide is highly similar to the sequence known from rabbit muscle but lacks a major part of its multiphosphorylation domain, including the main phosphorylation site for cAMP-dependent protein kinase (PKA). Analysis of this region by reverse-transcribed polymerase chain reaction (RT-PCR) in several human and rabbit tissues demonstrates that it is subject to elaborate differential mRNA splicing. Amino acids 1012-1024 of the full-length rabbit sequence, including the major PKA phosphorylation site, and amino acids 1025-1041, which harbor at least one endogenous phosphorylation site, can be deleted from the predicted polypeptide individually or in combination. Molecules lacking one or both of these segments constitute a major part of the alpha M subunit population in many rabbit tissues and constitute the vast majority in all human tissues analyzed. Similar, tissue-dependent differential splicing events could be detected by RT-PCR in the human alpha subunit isoform from liver (alpha L). The expression of the differentially spliced alpha M subtypes differs markedly between corresponding human and rabbit tissues. Sequence divergence in this region is particularly high, not only between the muscle and liver isoforms, but also between alpha M sequences from four different animal species. Moreover, a duplication of the exon encoding the main PKA phosphorylation site was discovered in the mouse. Thus, the multiphosphorylation domain of the phosphorylase kinase alpha subunit isoforms is subject to pronounced structural variation not only between different tissues of one organism via differential splicing, but also in the course of evolution.

Alternative Splicing↗
PubMed1976

[Correlation of active and inactive forms of phosphorylase and glycogen synthetase in the liver of the developing chick embryo].

The marked changes in the activity and ratio of the active and inactive forms of glycogen synthetase and phosphorylase were found in the liver of developing chick embryo. The activity of phosphorylase increased in the liver from the 8th day of incubation till the end of embryogenesis. The practically complete activation of the inactive form of this enzyme was noted in the end of embryogenesis. The activity of glycogen synthetase in the liver increased till the 17th day of incubation and sharply decreased more than 6 times thereafter. The maximum of the active form of glycogen synthetase (1/3 of total activity) was noted on the 17th day as well. The changes in the content of glycogen synthetase in the liver are synchronized with those in the activity and ratio of the active and inactive forms of glycogen synthetase. The role of changes in the concentration of glucose and glucose-6-phosphate in the control of ratio of the active and inactive forms of enzymes of glycogen metabolism is discussed.

Age Factors↗
PubMed1993

Characterization of the gene for rat phosphorylase kinase catalytic subunit.

Phosphorylase kinase, a key enzyme in glycogen metabolism, has a subunit composition of (alpha beta gamma delta)4, in which the alpha and beta subunits are regulatory, delta is calmodulin, and the gamma subunit is catalytic. As one segment of our studies on the regulation of the expression of phosphorylase kinase subunits, we present in this report the structure of the gene for the catalytic gamma subunit. The gene extends over 16 kilobase pairs (kb) of DNA, and contains eight introns within the coding region plus one 3.3-kb intron upstream in the 5'-untranslated region. Within this first intron, and also upstream of the transcription start site, are sequences homologous to defined regulatory elements, including some found in other muscle-specific genes. The positions of intron splice junctions for this gene have been compared with similar data for other protein kinase genes. A somewhat unexpected finding for the gamma subunit is that two of the splice junctions fall in the midst of highly conserved strings of amino acids, both of which have been nominally defined as functional domains for the protein kinases and appear to make key contributions to substrate binding and phosphotransferase catalysis.

Amino Acid Sequence↗
PubMed1993

A chimeric alpha-glucan phosphorylase of plant type L and H isozymes. Functional role of 78-residue insertion in type L isozyme.

Higher plant tissues such as potato tuber and leaf contain two alpha-glucan phosphorylase isozymes designated types L and H. Although the sequences of the two isozymes are highly conserved except for a 78-residue insertion found uniquely in the type L isozyme, they differ strikingly in affinities for substrates. To examine whether the insertion in the type L isozyme plays a role in enzymic functions, particularly in substrate specificities, we have constructed a chimeric enzyme, in which a 189-residue sequence of the type L isozyme including the insertion and its flanking regions is replaced by the corresponding sequence (112 residues) of the type H isozyme lacking the insertion. The gene for the chimeric enzyme as well as the cDNA for the type L isozyme were expressed at a low temperature in Escherichia coli cells under the control of the strong T7 RNA polymerase promoter. The purified chimeric phosphorylase was five times less active than the parent type L isozyme, but its affinity for glycogen was much higher than that of the type L isozyme and only slightly lower than that of the type H isozyme. The Michaelis constants of the chimeric enzyme for small oligosaccharides were comparable with those of the type L isozyme. These results provide evidence for the role of the 78-residue insertion in the type L isozyme, lowering the affinity of the enzyme for large, branched substrates probably through steric hindrance. It is also assumed that the corresponding region in the type H isozyme contains a high affinity site like the glycogen storage site occurring in the animal enzyme.

Amino Acid Sequence↗
PubMed1995

[Interaction of metal ions with the carboxyl group of Asp-5 in the active site of uridine phosphorylase from Escherichia coli K-12].

The effects of La3+ ions on enzymatic activity and difference absorption spectra of native and fluorescein isothiocyanate (FITC) modified uridine phosphorylase from E. coli K-12 have been studied. Excess La3+, unlike Ag+, only slightly decreases the enzyme activity but provokes similar changes in the absorption spectra of both native and modified proteins. The Kd value for La3+ ions (0.2 mM) coincides with that obtained earlier for Ag+. La3+ ions (0.2 mM) have no effect on the rate of the enzyme inactivation by diethylpyrocarbonate or tetranitromethane but increases the rate of its inactivation by Woodward's reagent K (WRK). Binding of La3+ (Kd = 0.2 mM) markedly decreases the thermal stability of the enzyme which increases with a further rise in the La3+ concentration. The values of Kd (0.2 mM) as well as the difference spectra and specific interactions with WRK indicate that one of the ligands interacting with metal ions is the carboxyl group of the Asp-5 residue. According to X-ray analysis data, this residue is involved in the formation of the active center of uridine phosphorylase.

Binding Sites↗
PubMedDate not supplied

[Exercise intolerance caused by muscular phosphorylase kinase deficiency. Contribution of in vivo metabolic studies].

A 33 year old man has been presenting since childhood an exertional muscle pain syndrome without myoglobinuria. Muscle biopsy revealed a vacuolar myopathy with glycogen excess in subsarcolemmal and intermyofibrillar spaces which was confirmed by electron microscopy. Plasma production of ammonia was abnormally high during exercise on a bicycle ergometer while the raise of lactate was normal. NMR spectroscopy showed an increased muscle glycogen content, with a slight and delayed drop of the pH during exercise. Phosphorylase b kinase activity was undetectable in muscle specimen whereas activities of others enzymes of carbohydrate metabolism were normal. Clinical presentation of our patient is compared to that of the reported cases of phosphorylase b kinase deficiency.

Adult↗
PubMed1977

[Purification and kinetic properties of glycogen phosphorylase A from rabbit liver].

The purification of phosphorylated form of rabbit liver glycogen phosphorylase (phosphorylase A) using the chromathography on a omega-amino-hexyl-Sepharose column has been carried out. The yield is about 90%, the specific activity is equal to 90 mkmol Pi/min. mg. The enzyme samples appeared essentially homogeneous when analyzed by polyacrylamide gel electrophoresis. The Km value of glucose-1-phosphate in the presence of AMP is 4--6 mM, which is higher than that found previously. When the glucose-1-phosphate concentration was varied, deviations from Michaelis-Menten kinetics were observed in the presence of glucose or ATP (Hill slopes of 1.6), but these deviations were virtually abolished by the presence of AMP. With glucose-1-phosphate as the substrate the Ki value of glucose is 57 mM in the absence of AMP and 150 mM in the presence of this effector. The Ki value of ATP is less dependent on the presence of AMP. The synergism in combined action of glucose and ATP has been revealed.

Adenosine Monophosphate↗
PubMed1996

Thermal inactivation of skeletal muscle phosphorylase b: protective effect of the specific ligands.

The kinetics of the thermal inactivation of rabbit skeletal muscle phosphorylase b have been studied. Under the condition used (0.08 M HEPES, pH 6.8, 75 mu g/ml phosphorylase b) the enzyme quickly loses its activity at the temperature higher then 49 degrees C. All the specific ligands tested (substrate -glucose 1-phosphate, competitive inhibitor - glucose, allosteric activator - AMP, and allosteric inhibitors - glucose 6-phosphate and FMN) have the pronounced protective effect. The strongest protective action have glucose 6-phosphate and FMN. Quantitative analysis of the inactivation rate constant versus ligand concentration curves allow the enzyme affinity for ligands to be characterized.

Adenosine Monophosphate↗
PubMed1977

Enzyme-synthetic approach to demonstration of phosphorylase activity in the living rabbit cornea.

An enzyme-synthetic method of demonstrating phosphorylase was applied to the living rabbit cornea, and polyglucose particles synthesized from glucose-1-phosphate in vivo were studied electron microscopically. In the corneas in which the medium for phosphorylase was applied from the anterior chamber or the bulbar subconjunctiva, synthesized polyglucose particles were found in the cytoplasmic matrices of the epithelium. When the medium was deposited in the conjunctival sac, a few synthesized polyglucose particles were found in the cytoplasmic matrices of only the superficial layer of the corneal epithelium. These findings suggest that metabolites for glycogen metabolism come mainly from the aqueous humor in the anterior chamber. The polyglucose particles synthesized by the enzyme-synthetic method in vivo resemble native glycogen particles. In addition, these particles were not overproduced because the synthesis of polyglucose is probably regulated in vivo.

Animals↗
PubMed1997

Synthesis of 6-aminouracils and pyrrolo[2,3-d]pyrimidine-2,4-diones and their inhibitory effect on thymidine phosphorylase.

Inhibitors of thymidine phosphorylase (dThdPase) are expected to suppress the growth and metastasis of tumor cells by inhibition of angiogenesis and were designed by utilizing the three dimensional structure of the enzyme. 5-Substituted 6-aminouracils (5) and 7-substituted pyrrolo[2,3-d]pyrimidine-2,4-diones (6) were synthesized and tested for inhibition of human placenta thymidine phosphorylase. 5-Bromo-6-aminouracil (5d), 5-cyano-6-[3-(methylamino)propyl]-uracil (5e), and 7-(2-aminoethyl)-pyrrolo[2,3-d]pyrimidine-2,4-dione (6c) inhibited dThdPase with IC50s of 7.6, 3.8 and 44.0 microM, respectively.

Antimetabolites, Antineoplastic↗
PubMed1987

The first step in the functional inactivation of the Escherichia coli polynucleotide phosphorylase messenger is a ribonuclease III processing at the 5' end.

The transcripts covering pnp, the gene encoding polynucleotide phosphorylase, are processed by ribonuclease III. In this study, it is shown that the steady state level of the pnp mRNA increased 11-fold in a ribonuclease III-deficient strain. The synthesis rate of this messenger is only slightly affected in the mutant strain whereas the half-life, which is 1.5 min in the wild type, is considerably increased to more than 40 min. Moreover, polynucleotide phosphorylase is 10-fold over-expressed in the mutant strain, which shows that unprocessed pnp mRNA is functional. The position of the ribonuclease III-sensitive site suggests that the sequence involved in the stabilization of the pnp mRNA is located at the 5' end of the message and that the RNase III processing triggers the decay of the transcripts downstream. A similar function for ribonuclease III in the processing of the messenger for the beta beta' subunits of RNA polymerase is proposed.

Endoribonucleases↗
PubMed1980

Isolation of a polynucleotide phosphorylase mutant using a kanamycin resistant determinant.

Insertion in an episome of a kanamycine-resistant element (Tn5) at the polynucleotide phosphorylase gene level, results, after transduction into a wild strain, by the loss of activities specific to polynucleotide phosphorylase. A low phosphorolytic activity is nevertheless detectable in crude extracts, but no longer in extracts slightly purified after heat treatment at 54 degrees C. The part played by other enzymes in these activities is discussed. Bacterial growth is not affected by introduction of the mutation.

Coliphages↗
PubMed1994

The gamma phosphorylase kinase gene, Phkg, maps to mouse chromosome 5 near Gus.

Phosphorylase kinase is a multimeric regulatory enzyme in the glycogenolytic pathway. Interest in various types of phosphorylase kinase enzyme deficiency has focused attention on cloning and mapping the enzyme subunits. We report the mapping of the catalytic gamma subunit gene, Phkg, to mouse Chromosome (Chr) 5 near beta-glucuronidase (Gus), between alpha fetoprotein (Afp) and erythropoietin (Epo). In addition, PCR-based polymorphism assays have been developed for the human (EPO) and mouse erythropoietin genes, and a unique recombinant inbred strain distribution pattern has been defined for Epo, a distal anchor marker on mouse Chr 5.

Alleles↗
PubMed2002

Comment on "The first description of an archaeal hemicellulase: the xylanase from Thermococcus zilligii strain AN1": evidence that the unique N-terminal sequence proposed comes from a maltodextrin phosphorylase.

Uhl and Daniel reported in this journal in 1999 (Extremophiles 3:263-267) the characterization of the first archaeal hemicellulase with a unique N-terminal sequence showing no homology with any xylanase or other protein from the databases. A genomic library of the chromosomal DNA of Thermococcus zilligii strain AN1 was screened by using a degenerate probe deduced from the N-terminal sequence. A positive clone was identified, and an amino acid sequence analysis revealed that the N-terminal sequence from this protein and the N-terminal sequence from the putative xylanase of T. zilligii were identical. However, the comparison of the amino acid sequence of the protein with sequences in the main protein databases revealed significant similarities with maltodextrin phosphorylases. In conclusion, it is likely that the N-terminal sequence proposed by Uhl and Daniel is not that of the T. zilligii xylanase, but corresponds to an archaeal T. zilligii maltodextrin phosphorylase.

Amino Acid Sequence↗