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At least 19 recordsLinked to original sources
PubMed1978

Kinetic analyses of the sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose enzyme II complex of the bacterial phosphotransferase system.

The sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose Enzyme II complex of the phosphotransferase system has been analyzed kinetically. Initial rates of phosphoryl transfer from glucose-6-P to methyl alpha-glucopyranoside were determined with butanol/urea-extracted membranes from Salmonella typhimurium strains. The kinetic mechanism was shown to be Bi-Bi Sequential, indicating that the Enzyme II possesses nonoverlapping binding sites for sugar and sugar phosphate. Binding of the two substrates appears to occur in a positively cooperative fashion. A mutant with a defective glucose Enzyme II was isolated which transported methyl alpha-glucoside and glucose with reduced maximal velocities and higher Km values. In vitro kinetic studies of the transphosphorylation reaction catalyzed by the mutant enzyme showed a decrease in maximal velocity and increases in the Km values for both the sugar and sugar phosphate substrates. These results are consistent with the conclusion that a single Enzyme II complex catalyzes both transport and transphosphorylation of its sugar substrates.

Biological Transport↗
PubMed1988

Identification and functional reconstitution of phosphate: sugar phosphate antiport of Staphylococcus aureus.

Resting cells of Staphylococcus aureus displayed a phosphate (Pi) exchange that was induced by growth with glucose 6-phosphate (G6P) or sn-glycerol 3-phosphate (G3P). Pi-loaded membrane vesicles from these cells accumulated 32Pi, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of 32Pi (and L-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (DL-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both Pi and DL-lactate to establish an internal pool of Pi. This trans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal Pi. Reconstitution of membrane protein allowed a quantitative analysis of Pi-linked exchange. Pi-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous 32Pi: Pi exchange (Kt's of 2.2 and 1.4 mM; Vmax's of 180 and 83 nmol Pi/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (Kt = 27 microM) over G3P (Kt = 1.3 mM) and Pi (Kt = 2.2 mM), suggesting that the same antiporter was induced in both cases. We conclude that 32Pi: Pi exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport, Active↗
PubMed1995

Allosteric activation of rabbit reticulocyte guanine nucleotide exchange factor activity by sugar phosphates and inositol phosphates.

Sugar phosphates are required to maintain active rates of translation in gel-filtered rabbit reticulocyte lysates. They may stimulate polypeptide chain initiation by acting as NADPH generators or by a direct interaction with initiation factor(s). We now provide evidence for the allosteric activation of the purified guanine nucleotide exchange factor (eIF-2B) by sugar phosphates and inositol phosphates. In the presence of microM fructose 1,6-bisphosphate, the rate of eIF-2B-catalyzed GDP/GTP exchange is increased approximately 2-fold. The half-maximal concentration for stimulation of eIF-2B activity (SC50) is 57 microM. The binding of GTP to isolated eIF-2B is stimulated 1.5-fold, whereas GTP-binding to ALP-treated eIF-2B is not affected by sugar phosphates. Inositol 1,4-bisphosphate, like fructose 1,6-bisphosphate, stimulates 2-3-fold the activity of the isolated eIF-2B (SC50, 140 microM).

Allosteric Regulation↗
PubMed1990

The effect of the progression of vitamin A deficiency on glucose, galactose and mannose incorporation into sugar phosphates and sugar nucleotides in hamster liver.

The incorporation of [2-3H]mannose into dolichyl phosphate mannose and glycoproteins is markedly reduced in livers of vitamin A-deficient hamsters. To determine whether vitamin A deficiency selectively alters the level of mannose incorporation into sugar phosphates and sugar nucleotides, we studied the in vivo incorporation of [2-3H]mannose, [5-3H]glucose, and [4,5-3H]galactose into sugar phosphates and sugar nucleotides. Male hamsters fed either a vitamin A-depleted or a retinoic acid-supplemented (3 micrograms/g) diet were used at 4, 6 and 8 wk of age; the animals were killed at various time points after an intraperitoneal injection of the radiolabeled sugar. A two- to threefold increase in the amount of [2-3H]mannose was found in liver of hamsters fed a vitamin A-depleted diet for 4 wk, resulting in enhanced incorporation into mannosyl-phosphate and guanosine diphosphate (GDP) mannose. As deficiency progressed, there was a smaller increase in [2-3H]mannose and a significant decrease in [3H]mannose-phosphate and GDP-[3H]mannose, suggesting a decreased mannose kinase activity. [5-3H]Glucose-labeled livers showed no difference in the total uptake of the label or its incorporation into uridine diphosphate glucose and galactose-phosphate during the 8-wk study. However, the synthesis of glucosyl-phosphate was reduced by 50 to 90% at 6 and 8 wk of deficiency, suggesting an impaired gluco-kinase activity. In hamsters injected with [4,5-3H]galactose only [3H]glucose was found within 5 min in the free sugar fraction. In contrast, as much as 70% of the label in the sugar phosphate and sugar nucleotide fraction remained as [3H]galactose even at 60 min. These effects on sugar, sugar phosphate and sugar nucleotide formation in part may explain the effects of vitamin A deficiency on glycoconjugate biosynthesis.

Animals↗
PubMed2003

A high-throughput solid-phase extraction assay capable of measuring diverse polyprenyl phosphate: sugar-1-phosphate transferases as exemplified by the WecA, MraY, and MurG proteins.

The bacterial proteins WecA and MraY are members of the polyprenyl phosphate:N-acetylhexosamine-1-phosphate transferase family, each of which catalyzes the transfer of a specific hexosamine 1-P from a soluble UDP-hexosamine substrate to a bactoprenyl phosphate carrier at the membrane surface. Currently, assays designed to quantitate the activity of these enzymes rely on paper chromatography or liquid-liquid extractions or are specialized to a few members of the family. We describe a generalizable, high-throughput, one-pot assay for these activities that uses a solid-liquid bead-based separation system to selectively adsorb the highly hydrophobic products of reaction. By judicious choice of radiolabeled UDP-hexosamine precursor, the same format can be used to quantitate not only diverse members of this transferase family, but also enzymes that catalyze the further modification of these transferase products. This possibility is exemplified by the MurG protein of bacterial cell wall synthesis, which catalyzes the addition of an N-acetylglucosamine residue to the product of the MraY reaction. Thus, the use of this flexible assay tool will allow a critical biochemical and enzymologic analysis of many such membrane-bound transferases in a similar setting.

Bacterial Outer Membrane Proteins↗
PubMed1983

Survey, purification, and properties of sugar phosphate phosphohydrolase among microorganisms.

Sugar phosphate phosphohydrolase was purified approximately 500- to 600-fold to apparent homogeneity from Escherichia coli B, Escherichia coli C, Escherichia coli var. communior, Escherichia acidilactici, Enterobacter aerogenes, Neisseria meningitidis, and Saccharomyces cereviseae. The molecular weights of the enzyme as estimated by gel filtration ranged from 97 X 10(3) to 101 X 10(3). The enzyme was composed of two subunits with the same molecular weight which ranged from 50 X 10(3) to 52 X 10(3), as determined by sodium dodecyl sulfate gel electrophoresis. Homogeneous enzyme preparations hydrolyse all the tested alpha-D-aldohexose 1-phosphate, D-(keto or aldo)hexose 6-phosphate, and pentose phosphate substrates significantly. When the microorganisms were transferred from growth medium with 1% glucose to that without glucose, there were dramatic increases in both the specific and total enzyme activities. At least three isozymes appeared to be present in S. cereviseae, and two appeared to be present in E. coli B, E. coli var. communior, and N. meningitidis. Rabbit antiserum immunized against sugar phosphate phosphohydrolase purified from E. coli B cross-reacted with both the crude extracts and purified preparations of the enzyme from the other microorganisms. The presence of neither sugar phosphate phosphohydrolase activity nor immunocrossreacting material was detected in the following microorganisms: Aspergillus niger, Azotobacter chroococcum, Bacillus subtilis, Bacillus pumilis, Citrobacter freundii, Clostridium butyricum, Corynebacterium xerosis, Flavobacterium aquatile, Flavobacterium synxanthum, Lactobacillus bulgaricus, Micrococcus coralinus, Neisseria perflava, Neurospora crassa, Penicilium expansum, Penicilium notatum, Proteus mirabilis, Proteus vulgaris, Pseudomonas fluorescens, Saccharomyces fermenti, Sarcina lutea, and Streptomyces antibioticus. At present, no conclusive relationship can be established between the phosphoenolpyruvate phosphotransferase system and the enzyme sugar phosphate phosphohydrolase among microorganisms. The physiological role of sugar phosphate phosphohydrolase as a transferase and regulatory enzyme is discussed.

Bacteria↗
PubMed1990

High-performance reversed-phase ion-pair chromatographic study of myo-inositol phosphates. Separation of myo-inositol phosphates, some common nucleotides and sugar phosphates.

A detailed study of all the major chromatographic variables affecting the retention behaviour and separation of myo-inositol phosphates in reversed-phase ion-pair chromatographic systems was carried out. The parameters studied included the eluent concentration of the pairing ion, the eluent concentration of the organic modifier and the buffer salt, the pH of the eluent, the minimum column plate count necessary for the separation of the inositol trisphosphate isomers and isocratic and gradient modes of separation. The retention behaviour of some common nucleotides and sugar phosphates was also investigated as these phosphates present chromatographic interference problems in biochemical studies based on the cellular incorporation of [32P]Pi. The separation methods developed appear to be superior to established anion-exchange separation techniques in terms of separation speed and "mildness" of the chromatographic conditions.

Chromatography, High Pressure Liquid↗
PubMed1986

Variable stoichiometry of phosphate-linked anion exchange in Streptococcus lactis: implications for the mechanism of sugar phosphate transport by bacteria.

Phosphate/2-deoxyglucose 6-phosphate antiport in Streptococcus lactis showed an exchange stoichiometry that varied over a 2-fold range when assay pH was shifted between pH 8.2 and pH 5.2. At pH 7.0 and above, 2 mol of phosphate moved per mol of sugar phosphate; at pH 6.1 the ratio was 1.5:1, while at pH 5.2 the overall stoichiometry fell to 1.1:1. This pattern was not affected by valinomycin in potassium-based media, nor could variable stoichiometry be attributed to altered hydrolysis of the sugar phosphate substrate. In kinetic studies at pH 7.0 or pH 5.2, sugar 6-phosphate was a competitive inhibitor of phosphate transport, indicating operation of a single system. Parallel tests showed that the affinity of antiport for its sugar 6-phosphate substrate was insensitive to pH in this range. Overall, such results suggest a neutral exchange that has specificity for monovalent phosphate but that selects randomly among the available mono- and divalent sugar 6-phosphates. A simple model that shows this behavior suggests a mechanistic role for anion exchange in bacterial transport of sugar phosphate or other organic anions.

Anions↗
PubMedDate not supplied

Competition of pyridoxal 5'-phosphate with ribulose 1,5-bisphosphate and effector sugar phosphates at the reaction centers of the spinach ribulose 1,5-bisphosphate carboxylase/oxygenase.

The stimulation of the carboxylase reaction by effectors of ribulose 1,5-bisphosphate carboxylase/oxygenase displays higher sensitivity towards pyridoxal 5'-pyridoxal 5'-phosphate inhibition than the catalytical process itself. Pyridoxal 5'-phosphate binding to the enzyme is not affected by the modulators 6-phosphogluconate and fructose 1,6-bisphosphate at low concentrations at which these agents stimulate the carboxylation rate. At higher concentrations these sugar phosphates protect the enzyme against pyridoxal 5'-phosphate inhibition in a similar fashion like the substrate ribulose 1,5-bisphosphate. Such protection experiments in combination with spectrophotometrical studies of pyridoxal 5'-phosphate binding demonstrate two binding states of ribulose 1,5-bisphosphate at the reaction centers of the enzyme with different requirements for Mg2+. 6-Phosphogluconate functions as protector only in the presence of Mg2+. Our results imply a competition between pyridoxal 5'-phosphate and substrate or effector sugar phosphates at the reaction centers of the spinach carboxylase. It is proposed that the pyridoxal 5'-phosphate inhibition of the stimulatory activity of these effectors originates from a modification of the regulatory sites of the enzyme caused by pyridoxal 5'-phosphate binding to the catalytical sites.

Binding Sites↗
PubMed1988

Thermodynamics of isomerization reactions involving sugar phosphates.

Thermodynamics of isomerization reactions involving sugar phosphates have been studied using heat-conduction microcalorimetry. For the process glucose 6-phosphate2-(aqueous) = fructose 6-phosphate2- (aqueous), K = 0.285 +/- 0.004, delta Go = 3.11 +/- 0.04 kJ.mol-1, delta Ho = 11.7 +/- 0.2 kJ.mol-1, and delta Cop = 44 +/- 11 J.mol-1.K-1 at 298.15 K. For the process mannose 6-phosphate2- (aqueous) = fructose 6-phosphate2- (aqueous), K = 0.99 +/- 0.05, delta Go = 0.025 +/- 0.13 kJ.mol-1, delta Ho = 8.46 +/- 0.2 kJ.mol-1, and delta Cop = 38 +/- 25 J.mol-1.K-1 at 298.15 K. The standard state is the hypothetical ideal solution of unit molality. An approximate result (-14 +/- 5 kJ.mol-1) was obtained for the enthalpy of isomerization of ribulose 5-phosphate (aqueous) to ribose 5-phosphate (aqueous). The data from the literature on isomerization reactions involving sugar phosphates have been summarized, adjusted to a common reference state, and examined for trends and relationships to each other and to other thermodynamic measurements. Estimates are made for thermochemical parameters to predict the state of equilibrium of the several isomerizations considered herein.

Calorimetry↗
PubMed1993

A rapid spectrophotometric estimation of individual phosphates and phosphonates: its application to chromatography of sugar phosphates and their phosphonate analogues.

A compound containing the -PO3H2 group (phosphoric acid, one of its monoesters, or an alkylphosphonic acid) may be rapidly assayed by the decrease it produces in the absorbance at 450 nm of a buffered acidic solution of Fe3+ and N(-3) [corrected]. The method has been used to follow chromatograms of sugar phosphates and their phosphonomethyl analogues.

Chromatography, Ion Exchange↗
PubMed1990

Regulation of carbon flux from amino acids into sugar phosphates in Xenopus embryos.

Xenopus laevis oocytes and embryos are glycogenic cells, metabolizing sugar phosphates into glycogen. These cells have very low pyruvate kinase activity in vivo and, consequently, make little pyruvate and lactate through glycolysis. Nevertheless, oocytes and embryos do contain significant pyruvate and lactate levels. To determine the source of carbon for sugar phosphates and pyruvate, 14C-labeled intermediary metabolites were injected into fertilized eggs and their metabolism examined by thin-layer chromatography. Alanine, pyruvate, and lactate form a pool of carbon that fluxes into sugar phosphates. Cytosolic (nonmitochondrial) aspartate, oxaloacetate, and malate form a pool of carbon which is largely blocked in the short-term from entering the smaller alanine/pyruvate/lactate pool. The data indicate that the major source of carbon for sugar phosphates in fertilized eggs and rapidly cleaving embryos is the alanine/pyruvate/lactate pool. Pyruvate from this pool is converted in the mitochondria to phosphoenolpyruvate, which in turn is metabolized outside the mitochondria to sugar phosphates. A key enzyme in regulating flux from amino acid carbon to pyruvate is malic enzyme. Three malic enzyme isozymes, one soluble and two mitochondrial, were partially isolated and kinetically characterized from total ovarian tissue. Full-grown oocytes and eggs, however, have very low soluble malic enzyme activity, which results in the separation of the cytosolic aspartate/oxaloacetate/malate and alanine/pyruvate/lactate pools.

Amino Acids↗