The diversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate from the 2-C-methyl-D-erythritol 4-phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana.

2-C-Methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) is an intermediate of the plastid-localized 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co-factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds-3 mutant, defective in the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2-C-methyl-D-erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds-3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds-3 mutant also showed enhanced resistance to the phloem-feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP-mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds-3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.

Fully automated on-line two-dimensional liquid chromatography in combination with ESI MS/MS detection for quantification of sugar phosphates in yeast cell extracts.

A mass spectrometric quantitative assay was developed for the analysis of 10 sugar phosphates in the yeast Pichia pastoris. As a novelty, two-dimensional chromatography based on a fully automated heart-cutting LC-LC technique was introduced. Using a ten-port valve, ten fractions of the first chromatographic dimension, i.e. anion exchange chromatography (AEC), were transferred and separated by the orthogonal second dimension, i.e. separation on porous graphitized carbon. The chromatographic separation on the second dimension was optimized for each transferred fraction minimizing the separation time and ensuring complete removal of the salt constituents of the AEC eluents. The latter being crucial for electrospray mass spectrometric detection was confirmed by combining the LC-LC separation with on-line ICP-MS detection. These measurements showed that sodium elution was completed after 0.8 min. Consequently, an analysis time of 1 min per transferred peak was established. In this way, the excellent peak capacity given by ion exchange could be conserved in the second dimension at the same time enabling mass spectrometric detection. Sub-µM limits of detection could be obtained by the new LC-LC-MS/MS methods ranging between 0.03 and 0.19 µM for the investigated compounds (only 3GAP showed a LOD of 1 µM). The method was applied to the quantification of ten sugar phosphates in yeast extracts utilizing internal standardization with a fully labeled (13)C yeast extract. Typically, the standard uncertainties for N = 3 replicates assessed by the LC-LC-MS/MS set-up were <5%.

Selectivity issues in targeted metabolomics: Separation of phosphorylated carbohydrate isomers by mixed-mode hydrophilic interaction/weak anion exchange chromatography.

Phosphorylated carbohydrates are important intracellular metabolites and thus of prime interest in metabolomics research. Complications in their analysis arise from the existence of structural isomers that do have similar fragmentation patterns in MS/MS and are hard to resolve chromatographically. Herein, we present selective methods for the liquid chromatographic separation of sugar phosphates, such as hexose and pentose phosphates, 2- and 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, as well as glucosamine 1- and 6-phosphate utilizing mixed-mode chromatography with reversed-phase/weak anion-exchangers and a charged aerosol detector. The best results were obtained when the reversed-phase/weak anion-exchanger column was operated under hydrophilic interaction liquid chromatography elution conditions. The effects of various chromatographic parameters were examined and are discussed on the basis of a simple stoichiometric displacement model for explaining ion-exchange processes. Employed acidic conditions have led to the complete separation of α- and ß-anomers of glucose 6-phosphate at low temperature. The anomers coeluted in a single peak at elevated temperatures (>40°C) (peak coalescence), while at intermediate temperatures on-column interconversion with a plateau in-between resolved anomer peaks was observed with apparent reaction rate constants between 0.1 and 27.8×10(-4) s(-1). Dynamic HPLC under specified conditions enabled to investigate mutarotation of phosphorylated carbohydrates, their interconversion kinetics, and energy barriers for interconversion. A complex mixture of six hexose phosphate structural isomers could be resolved almost completely.

Determination of trehalose-6-phosphate in Arabidopsis seedlings by successive extractions followed by anion exchange chromatography-mass spectrometry.

A method for the detection of trehalose-6-phosphate (T6P) in tissue of the model plant Arabidopsis thaliana is presented. Liquid-liquid extraction (LLE) and mixed mode solid-phase extraction (SPE) were used for sample pretreatment followed by anion exchange chromatography (AEC) coupled with electrospray ionization mass spectrometry (MS) for highly selective quantitative analysis. LLE of plant material was performed with chloroform/acetonitrile/water (3:7:16, v/v/v) followed by SPE with Oasis MAX material, which significantly reduced the complexity of the extracts. On-line coupling of MS with gradient AEC using a sodium hydroxide eluent was accomplished with a postcolumn ion suppressor. The method allows specific quantification of T6P with good linearity for spiked plant extracts, from 80 nM to 1.3 microM (r(2)>0.98). The limit of detection in plant extracts was 40 nM. The recovery of the method was above 80% for relevant T6P levels. The method was applied to the determination of T6P in seedlings from four mutant A. thaliana lines (TRR1-4) resisting growth arrest caused by external supply of trehalose. Results reveal that T6P accumulation differed substantially in the four mutant lines and wild type (WT). It is concluded that the mutants circumvent the growth arrest observed in WT seedlings on 100mM trehalose by different mechanisms.

Erythrocyte galactose 1-phosphate quantified by isotope-dilution gas chromatography-mass spectrometry.

BACKGROUND: Measurements of alpha-D-galactose 1-phosphate (Gal-1-P) in erythrocytes are used to monitor the adequacy of dietary therapy in the treatment of galactosemia. We have devised a gas chromatography-mass spectrometry (GC/MS) isotope-dilution method for quantification of Gal-1-P. METHODS: We prepared trimethylsilyl (TMS) derivatives and used alpha-D-[2-(13)C]Gal-1-P as the internal standard for GC/MS. Results obtained with this method were compared with those determined by the established enzymatic method for samples from 23 healthy individuals (11 children and 12 adults), 9 suspected patients with galactosemia, 12 galactosemic patients on diet therapy, and 2 newly diagnosed toxic neonates. RESULTS: The method was linear up to 2.5 mmol/L with a lower limit of detection of 2.1 nmol (0.55 mg/L). Intra- and interassay imprecision (CVs) was 2.2-8.8%. In the 23 healthy individuals, values ranged from nondetectable to 9.2 micromol/L (2.4 mg/L of packed erythrocytes). Galactosemic patients on diet therapy had values of 10.9-45 mg/L of packed erythrocytes, whereas the newly identified patients had values of 166 and 373 mg/L. CONCLUSIONS: The GC/MS method is precise and useful over the wide range of concentrations needed to assess the galactose burden in patients with galactosemia.

Chemical structure and immunoreactivity of the lipopolysaccharide of the deep rough mutant I-69 Rd-/b+ of Haemophilus influenzae.

From the lipopolysaccharide of the deep rough mutant I-69 Rd--/b+ of Haemophilus influenzae two oligosaccharides were obtained after de-O-acylation and separation by high-performance anion exchange chromatography. Their chemical structures were determined by one- and two-dimensional 1H-, 13C- and 31P-NMR spectroscopy as alphaKdo-4P-(2-->6)-betaGlcN-4P-(1-->6)-alphaGlcN-1P and alphaKdo-5P-(2-->6)-betaGlcN-4P-(1-->6)-alphaGlcN-1P. The specificity of mAbs S42-21 and S42-16 specific for Kdo-4P or Kdo-5P, respectively [Rozalski, A., Brade L., Kosma P., Moxon R., Kusumoto S., & Brade H. (1997). Mol. Microbiol. 23, 569--577] was confirmed with neoglycoconjugates obtained by conjugation of the isolated oligosaccharides to BSA. In addition, a mAb S42-10-8 with unknown epitope specificity could be assigned using the neoglycoconjugates described herein. This mAb binds to an epitope composed of the bisphosphorylated glucosamine backbone of lipid A and Kdo-4P, whereby the latter determines the specificity strictly by the position of the phosphate group.

Probing the stereochemistry of E. coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (phenylalanine-sensitive)-catalyzed synthesis of KDO 8-P analogues.

The five-carbon phosphorylated monosaccharide analogues, D-arabinose 5-phosphate, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate, were separately condensed with (Z)- and (E)-[3-(2)H]-phosphoenolpyruvate (PEP) in the presence of Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (phe) to give in the case of (Z)-[3-(2)H]-PEP (3S)-[3-(2)H]-3-deoxy-D-manno-octulosonate 8-phosphate, (3S)-[3-(2)H]-3-deoxy-D-altro-octulosonate 8-phosphate, and (3S)-[3-(2)H]-3,5-dideoxy-D-altro-octulosonate 8-phosphate, respectively, whereas incubation with (E)-[3-(2)H]-PEP gives the corresponding (3R)-monosaccharides. These results are in complete agreement with the observed facial selectivity of DAH 7-P synthase for its normal substrates D-erythrose 4-phosphate and PEP and provide direct evidence that DAH 7-P synthase (phe) catalyzes the si face addition of the C3 of PEP to the re face of C1 of the phosphorylated monosaccharides tested. Products formed by DAH 7-P synthase (phe)-catalyzed condensation of (Z)- and (E)-[3-F]-PEP with E 4-P were completely characterized by (1)H and (19)F NMR analysis for the first time. Results of our studies suggest that disappearence of the double bond between C2 and C3 of PEP and formation of a bond between C3 of PEP and C1 of the phosphorylated monosaccharide tested occur in concert during the DAH 7-P synthase-catalyzed condensation reaction.

Trehalose 6-phosphate production with energy coupling fermentation by yeast cells.

We tried a method for the production of trehalose 6-phosphate (T6P) with energy-coupling fermentation by baker's yeast. T6P was produced in a reaction mixture containing glucose, 5'-UMP, MgSO4, inorganic phosphate, and dried cells of baker's yeast as the enzyme preparation, T6P was isolated from the reaction mixture and identified by TLC, HPLC, GC-MS, and enzymatic methods. The reaction conditions suitable for T6P production were investigated. The formation of T6P and its precursors, glucose 6-phosphate and UDPglucose, at various pHs and concentrations of substrates was examined. Accumulation of T6P was maximum with a reaction mixture containing 1 M glucose, 20 mM 5'-UMP, 20 mM MgSO4, 400 mM sodium phosphate buffer (pH 6.2), and 100 mg/ml dried cells of baker's yeast shaken at 37 degrees C for 6 h. The yield of T6P as a percentage of glucose was 11% (mol/mol) under these reaction conditions.

Purification and structural characterization of a filamentous, mucin-like proteophosphoglycan secreted by Leishmania parasites.

Parasitic protozoa of the genus Leishmania secrete a filamentous macromolecule that forms networks and appears to be associated with cell aggregation. We report here the purification of this parasite antigen from Leishmania major culture supernatant and its compositional (75.6% carbohydrate, 20% phosphate, 4.4% amino acids, w/w), structural, and ultrastructural characterization as a highly unusual proteophosphoglycan (PPG). Mild acid hydrolysis, which cleaves preferentially hexose 1-phosphate bonds, releases the PPG glycans. Their structures are Galbeta1-4Man, Manalpha1-2Man, Galbeta1-3Galbeta1-4Man, PO4-6(Galbeta1-3)0-2Galbeta1-4Man, and PO4-6(Arabeta1-2Galbeta1-3)Galbeta1-4Man. These glycans are also components of the parasite glycolipid lipophosphoglycan, but their relative abundance and structural organization in PPG are different. Some of them represent novel forms of protein glycosylation. 31P NMR on native PPG demonstrates that phosphate is exclusively in phosphodiester bonds and that the basic structure R-Manalpha1-PO4-6-Gal-R connects the glycans. A phosphodiester linkage to phosphoserine (most likely R-Manalpha1-PO4-Ser) anchors the PPG oligosaccharides to the polypeptide. PPG has a unique amino acid composition; glycosylated phosphoserine (>43 mol %), serine, alanine, and proline account for more than 87 mol % and appear to be clustered in large proteinase-resistant domains. Electron microscopy of purified PPG reveals cable-like, flexible, long (to 6 microm), and unbranched filaments. The overall structure of PPG shows many similarities to mammalian mucins. Potential functions of this novel mucin-like molecule for the parasites are discussed.

Separation of phosphorylated sugars using capillary electrophoresis with indirect photometric detection.

Adenosine monophosphate (AMP) and naphthalene disulfonate (NDS) have been characterized as electrolytes for the indirect photometric detection of phosphorylated sugars and other organophosphorus compounds of biochemical interest. This work has resulted in the CE separation on an uncoated capillary using 5 mM AMP and 100 mM boric acid at pH 7.2 of six metabolites (glucose-6-phosphate [G6P], fructose-6-phosphate [F6P]), fructose-1,6-bisphosphate [F-1,6-P], dihydroxyacetone phosphate [DHAP], glyceraldehyde-3-phosphate [G3P], and 2-phosphoglycerate [2-PG] or 3-phosphoglycerate [3-PG]) found in the glycolytic pathway. The detection limits using a 5-sec injection time were between 0.5 and 1 mg/L for these compounds, with the exception of G3P. Resolution between 3-PG and 2-PG is possible by the addition of magnesium ion, although the separation time is longer. A successful separation of five monophosphorylated sugars (G6P, F6P, ribose-5-phosphate [R5P], sucrose-6-phosphate [S6P], and 2-PG) has been performed using the same conditions as for the glycolytic pathway separation. A separation of bisphosphorylated sugars (glucose-1,6-bisphosphate [G-1,6-P],F-1,6-P, ribulose-1,5-bisphosphate [Ru-1, 5P], and sedoheptulose-1,7-bisphosphate [S-1, 7P]) could not be performed with AMP unless magnesium chloride was added. With NDS, a separation of these bisphosphorylated sugars can be obtained without the addition of magnesium chloride.

Structures of decasaccharide and tridecasaccharide tetraphosphates isolated by strong alkaline degradation of O-deacylated lipopolysaccharide of Pseudomonas fluorescens strain ATCC 49271.

Mild hydrazinolysis of Pseudomonas fluorescens strain ATCC 49271 lipopolysaccharide (LPS) followed by strong alkaline degradation and purification by anion-exchange HPLC resulted in two phosphorylated oligosaccharides (1 and 2). On the basis of compositional analysis and 1H, 13C, and 31P NMR spectroscopy, including 2D correlation spectroscopy (COSY), 2D rotating frame NOE spectroscopy (ROESY), and 2D inverse mode H-detected heteronuclear 1H-13C and 1H-31P correlation spectroscopy, the following two structures (1 and 2) could be identified [formula: see text] where Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-octulosonic acid, Non is 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonulosonic acid, and P is phosphate. Decasaccharide 1 and tridecasaccharide 2 represent an incomplete core and the complete core carrying one O-antigen repeating unit, respectively. Both are attached to the lipid A backbone but, due to their degradation protocol, they lack N- and O-acyl substituents, including N- and O-acetyl groups, the 5-N-acetimidoyl group of Non, the 2-N-alanyl group of GalN, and the 7-O-carbamoyl group of Hep as well as diphosphate, triphosphate, and, probably, some of the monophosphate groups that are present in the intact core oligosaccharide.

High-performance liquid chromatographic system for separating sugar phosphates and other intermediary metabolites.

A simple method for separating intermediates of carbohydrate metabolism, including the difficult-to-resolve sugar phosphates, using anion-exchange high-performance liquid chromatography is described. A gradient of decreasing borate concentration (10 to 0 mM) and increasing ionic strength (0 to 150 mM NH4Cl) separates phosphorylated sugars based on the strength of the ester complex that they form with borate anion. Lyophillized samples are reconstituted in a low ionic strength aqueous medium (5 mM triethanolamine-HCl, pH 8.1) and chromatographed on a commercially available anion-exchange column (Hamilton PRP-X100). The process requires only 3 h and permits nanomole detection of the phosphorylated intermediates of the glycolytic and pentose shunt pathways.

Separation and analysis of 4'-epimeric UDP-sugars, nucleotides, and sugar phosphates by anion-exchange high-performance liquid chromatography with conductimetric detection.

An anion-exchange HPLC method coupled with conductimetric detection was developed for the analysis of UDP-sugars, nucleotides, and sugar phosphates, each in a single chromatographic run. The analysis was applicable to concentrations over 50 pmol. The utility of this technique was demonstrated by the measurement of UDP-sugar 4'-epimerase activity of cell-free extracts from wild-type and mutant Neisseria meningitidis serogroup B strains. Additionally, the method has been applied to the analysis of the intermediates of glycolysis in human erythrocytes.

Phosphoenolpyruvate-dependent maltose:phosphotransferase activity in Fusobacterium mortiferum ATCC 25557: specificity, inducibility, and product analysis.

Phosphoenolypyruvate-dependent maltose:phosphotransferase activity was induced in cells of Fusobacterium mortiferum ATCC 25557 during growth on maltose. The disaccharide was rapidly metabolized by washed cells maintained under anaerobic conditions, but fermentation ceased immediately upon exposure of the cell suspension to air. Coincidentally, high levels of a phosphorylated derivative accumulated within the cells. Chemical and enzymatic analyses, in conjunction with data from 1H, 13C, and 31P nuclear magnetic resonance spectroscopy, established the structure of the purified compound as 6-O-phosphoryl-alpha-D-glucopyranosyl-(1-4)-D-glucose (maltose 6-phosphate). A method for the preparation of substrate amounts of this commercially unavailable disaccharide phosphate is described. Permeabilized cells of F. mortiferum catalyzed the phosphoenolpyruvate-dependent phosphorylation of maltose under aerobic conditions. However, the hydrolysis of maltose 6-phosphate (to glucose 6-phosphate and glucose) by permeabilized cells or cell-free preparations required either an anaerobic environment or addition of dithiothreitol to aerobic reaction mixtures. The first step in dissimilation of the phosphorylated disaccharide appears to be catalyzed by an oxygen-sensitive maltose 6-phosphate hydrolase. Cells of F. mortiferum, grown previously on maltose, fermented a variety of alpha-linked glucosides, including maltose, turanose, palatinose, maltitol, alpha-methylglucoside, trehalose, and isomaltose. Conversely, cells grown on the separate alpha-glucosides also metabolized maltose. For this anaerobic pathogen, we suggest that the maltose:phosphotransferase and maltose 6-phosphate hydrolase catalyze the phosphorylative translocation and cleavage not only of maltose but also of structurally analogous alpha-linked glucosides.

Chiroinositol deficiency and insulin resistance. III. Acute glycogenic and hypoglycemic effects of two inositol phosphoglycan insulin mediators in normal and streptozotocin-diabetic rats in vivo.

Two insulin mediators, inositol phosphoglycans, were isolated from bovine liver by methods previously developed for rat liver, i.e. chromatography on an AG 1 x 8 ion exchange column and selective elution with HCl at pH 2.0 and 1.3. The pH 2.0 mediator containing D-chiroinositol stimulated pyruvate dehydrogenase phosphatase, whereas the pH 1.3 mediator containing myo-inositol inhibited cAMP-dependent protein kinase. Each mediator was further purified by thin layer and Bio-Gel P4 column chromatography and injected ip into normal fed rats together with [U-14C]glucose. After 2.5 h, diaphragms were removed, and glycogen isolated. Insulin mediators, like insulin, stimulated [U-14C]glucose incorporation into glycogen by 150-160% in a dose-dependent manner in the nanomolar range. Mediators injected iv in the nanomolar range into low dose streptozotocin-diabetic rats decreased plasma glucose 30-45% in 30-60 min, with a return to basal concentrations after 150-180 min. These in vivo insulin-like effects of mediator were observed without changes in serum insulin concentrations. The pH 2.0 mediator was 50-100 times more active (per nmol organic phosphate) than the pH 1.3 mediator in the ip diaphragm glycogenesis assay. Mediator effects on diaphragm were completely blocked by preincubation with an immunopurified inositol phosphoglycan antibody. Both mediators were equally active iv in lowering plasma glucose (per nmol inositol) at concentrations comparable to those of insulin.

Glycogen synthesis in rat liver from a pool of free glucose.

Glycogen synthesis in isolated perfused livers or livers of anesthesized rats (in situ), was studied using radioactively labelled fructose, lactate, and inositol as substrates. The specific radioactivity of glucose and glycogen was measured at various times and compared with that of some intermediates. The results suggest that liver glycogen is formed from the pool of free glucose which in turn is fed by the so-called "direct and indirect pathway" of glycogen synthesis. This points to an important role of glucose-6-phosphatase, an enzyme complex subject to regulation by glucocorticoids, well known promoters of hepatic glycogen synthesis.

Highly purified lipid X is devoid of immunostimulatory activity. Isolation and characterization of immunostimulating contaminants in a batch of synthetic lipid X.

Lipid X, an early precursor in the biosynthesis of lipid A has been reported to directly induce cytokine release in macrophages but also to inhibit endotoxin-induced tumor necrosis factor (TNF) induction. In this report we provide evidence that these conflicting results could be due to contaminants present in different batches of lipid X used. Thus, in an apparently pure batch of crystalline lipid X as obtained by a published procedure (Macher, I. (1987) Carbohydr. Res. 262, 79-84) small amounts of N,O-acylated disaccharide-1-phosphates could be identified. Their isolation was achieved by gel filtration on Sephadex LH-20 and further analysis of fractions showing elevated limulus amebocyte lysate values by thin layer chromatography and reverse-phase high performance liquid chromatography (HPLC) in combination with bioassays. Identification of immunostimulatory by-products was possible by testing HPLC-fractions for TNF-induction in bone marrow-derived mouse macrophages. Applying these procedures a disaccharide-1-phosphate, containing four 3(R)-hydroxymyristic acids at positions 2, 3, 2', 3', was identified as the main immunostimulatory side product. Two isomeric hydrolysis products of this compound with only three 3(R)-hydroxymyristic acid moieties attached to the disaccharide-1-phosphate were also identified. Surprisingly, these compounds behave quite differently in the TNF induction test. The disaccharide-1-phosphate, acylated at positions 2, 2', 3', is a very potent inducer of TNF-release whereas the corresponding isomer containing the 3(R)-hydroxymyristic acids in positions 2, 3, 2', does not induce TNF release, but strongly inhibits TNF release as induced by the former compound. Thus, contamination of "pure" lipid X with immunostimulatory or immunoinhibitory impurities may explain the divergent pharmacological profiles which were attributed to synthetic lipid X.

Thin-layer electrophoretic separation of monosaccharides, oligosaccharides and related compounds on reverse phase silica gel.

The electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcohols and sugar acids were determined in 0.3 M borate buffer, pH 10, using thin-layer electrophoresis on silanized silica gel, pretreated with octanol-1. A rapid separation of a number of sugars, occurring in foods, could be achieved. Using a 0.05-0.1 M neutral solution of barium acetate as electrolyte, thin-layer electrophoresis allowed excellent and rapid separation as well as identification of all common uronic acids which are constituents of many acidic polysaccharides.