Control of CO2 fixation regulation of stromal fructose-1,6-bisphosphatase in spinach by pH and Mg2+ concentration

The effect of pH and of Mg²⁺ concentration on the light activated form of stromal fructose-1,6-bisphosphatase (FBPase) was studied using the enzyme rapidly extracted from illuminated spinach chloroplasts. The (fructose-1,6-bisphosphate^4-)(Mg²⁺) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg²⁺ concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg²⁺ dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg²⁺ concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg²⁺ concentrations indicates, that the effect is not a consequence of the pH and Mg²⁺ concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate^3- (uncomplexed), resulting in a change of the activation state of the enzyme. The interplay between a rapid effect on the concentration of the substrate and a delayed effect on the activation state enables a rigid control of stromal FBPase by stromal Mg²⁺ concentrations and pH. Fructose-1,6-bisphosphatase is allosterically inhibited by fructose-6-phosphate in a sigmoidal fashion, allowing a fine control of the enzyme by its product.Abbreviations Fru1,6 bis P fructose-1,6-bisphosphate - Fru6P fructose-6-phosphate - FBPase fructose-1,6-bisphosphatase Some of these results have been included in a preliminary report (Heldt et al. 1984)     

Regulation of fructose-1,6-bisphosphatase activity in leaves

Fructose-1,6-bisphosphatase (EC 3.1.3.11) activity increased markedly (greater than 10-fold) upon illumination of wheat leaves. Darkening caused a relatively slow but complete reversal of light activation. The effects of O₂ and CO₂ concentration and light intensity on fructose-bisphosphatase activation were measured. In ratelimiting light, 2% O₂ stimulated enzyme activity, whereas varying the CO₂ concentration had little effect. In saturating light, lowering the oxygen tension had no effect, but CO₂ at near-saturating concentrations for photosynthesis inhibited enzyme activity. Dark inactivation of the enzyme was completely prevented by incubation of leaves in N₂, but was facilitated by O₂, indicating that O₂ is the major oxidant in darkened leaves. It is argued that while fructose bisphosphatase is redox-regulated in leaves, modulation of enzyme activity by this mechanism is unlikely to contribute to the regulation of CO₂ fixation in leaves.     

Inhibited light activation of fructose and sedoheptulose bisphosphatase in spinach chloroplasts exposed to osmotic stress

The light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) was inhibited in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Decreases in the velocity and magnitude of light activation correlated with the overall reduction in CO₂ fixation rates. Responses of osmotically stressed chloroplasts to both varying pH and exogeous dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerete (PGA) were examined. In the presence of DHAP, the absolute rate of CO₂ fixation was increased and this increase was most pronounced at alkaline pH. Enhanced light activation of these enzymes was also observed under these conditions. However, in the presence of PGA, similar increases in photosynthetic rate and enzyme activation were not evident. Light-dependent stromal alkalization was unaffected by the stress treatments. Inhibition of light activation under hypertonic conditions is discussed in terms of substrate availability, possible alterations of the redox state of ferredoxin and associated electron carriers, and inhibited enzyme-enzyme or enzyme-substrate interactions involved in the light activation process.Abbreviations and symbols DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - _s osmotic potential     

Regulation of fructose-1,6-bisphosphatase activity in intact chloroplasts. Studies of the mechanism of inactivation

1. The aim of this work was to investigate the mechanism of dark inactivation of fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) in isolated intact chloroplasts of Triticum aestivum.

2. Dark inactivation of the enzyme, which was rapid under aerobic conditions, was prevented under anaerobic conditions when chloroplasts were incubated in the absence of an electron acceptor. Electron acceptors such as oxaloacetate readily brought about inactivation under anaerobic conditions whether chloroplasts were illuminated or in the dark. Inactivation of the enzyme also occurred if illuminated or darkened anaerobic chloroplasts were exposed to oxygen.

3. Pyocyanine, which catalyses a cyclic electron flow around Photosystem I, also caused inactivation of the enzyme in illuminated, anaerobic chloroplasts.

4. It is proposed that the activity of fructose-1,6-bisphosphatase is regulated by the availability of electrons, and thus by electron acceptors, and that dark inactivation may occur by a direct reversal of the activation process.     

Purification and properties of pea (Pisum sativum L.) thioredoxin f,a plant thioredoxin with unique features in the activation of chloroplast fructose-1,6-bisphosphatase

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (M_r) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an M_r of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at — 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP⁺-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa M_r = 6324 M^–1 · cm^–1). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.Abbreviations DDT dithiothreitol - ELISA enzyme-linked immunosorbent assay - FBPase fructose- 1,6-bisphosphatase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Td thioredoxin The authors are grateful to Mrs. Francisca Castro and Mr. Narciso Algaba for skilful technical assistance. This work was supported by grant PB87-0431 of Dirección General de Investigación Cientifica y Técnica (DGICYT, Spain).     

Activation of chicken liver fructose-1,6-bisphosphatase by oxidized glutathione

Treatment of chicken liver fructose-1,6-bisphosphatase with oxidized glutathione (GSSG) leads to an increase in activity. This activation is markedly enhanced if treatment is performed in the presence of AMP or Mn²⁺. The effects of AMP and Mn²⁺ appear to be synergistic. The maximal activation is over 13-fold and is accompanied by the disappearance of 4 sulfhydryl groups per molecule of enzyme. Both fructose 1,6-bisphosphate and fructose 2,6-bisphosphate can largely prevent this activation. Activation can be reversed by dithiothreitol or cysteine. It appears that GSSG activates this enzyme by thiol/disulfide exchanges with the enzyme's specific sulfhydryl groups.     

Overexpression of the potential herbicide target sedoheptulose-1,7-bisphosphatase from Spinacia oleracea in transgenic tobacco

Several proteins are recalcitrant to expression in Escherichiacoli. To explore transgenic plants as an alternative expressionsystem, the gene encoding the potential herbicide target sedoheptulose-1,7-bisphosphatase (SBPase, EC 3.1.3.37) was expressed in transgenic tobacco(Nicotiana tabaccum) under the control of a duplicatedCaMV 35S RNA promoter. The active protein, a key enzyme in the Calvin cycle,accumulated to approximately 1.2% of total soluble protein. In order to purifyrecombinant SBPase, a sequence encoding six histidine residues was insertedC-terminally which allows a one step purification via Ni²⁺-NTAaffinity chromatography. N-terminal amino acid sequence analysis of the purifiedprotein confirmed processing of the transit peptide and revealed the previouslyunknown cleavage site. The transit peptide consists of 67 amino acids followedby the mature SBPase subunit of 342 amino acids including the C-terminalfusion. Purified SBPase was found to be enzymatically active after reduction with DTTand showed many biochemical properties of the native enzyme such as thedependence on Mg²⁺ and a pH optimum of 8.3. Subsequently, SBPaseproduced in transgenic tobacco was used in large-scale screening for thediscovery of novel herbicides.     

Co-operative activation of chloroplast fructose-1,6-bisphosphatase by reductant,pH and substrate

Intact chloroplasts capable of high rates of photosynthesis fail to reduce CO₂ when illuminated in the absence of oxygen. While anaerobiosis limits proton gradient formation leading to ATP deficiency (Ziem-Hanck, U. and Heber, U. (1980) Biochim. Biophys. Acta 591, 266–274), light activation of fructose-1,6-bisphosphatase was also inhibited by anaerobiosis, whereas light activation of NADP-malate dehydrogenase was stimulated by anaerobiosis, indicating that reductant was still available for light activation. The chloroplast pool of NADP was largely reduced during illumination under anaerobiosis and electron transport to oxaloacetate was not inhibited by anaerobic conditions. Significant light activation of fructose-bisphosphatase was observed in anaerobic chloroplasts with 3-phosphoglycerate as substrate, but not with dihydroxyacetone phosphate (3-phosphoglycerate supports electron transport and hence proton gradient formation). In the absence of added substrates, illumination of anaerobic chloroplasts resulted in some light activation of fructose-bisphosphatase when the pH of the medium was increased. Under these conditions, light activation was stimulated by dihydroxyacetone phosphate. Dihydroxyacetone phosphate added together with oxaloacetate allowed light activation of fructose-bisphosphatase in anaerobic chloroplasts, while neither substrate added alone was effective. Formation of a transthylakoid proton gradient can therefore substitute for an alkaline suspension medium by causing an alkaline shift of the stromal pH on illumination. The data are interpreted as indicating that fructose-bisphosphatase, but not NADP-malate dehydrogenase, requires an alkaline pH and the presence of substrate for rapid reductive light activation and they bear on the interpretation of the lag observed in photosynthesis in chloroplasts and leaves on illumination after a prolonged dark period.     

Biological evaluation and SAR analysis of novel covalent inhibitors against fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase (FBPase) is an attractive target for affecting the GNG pathway. In our previous study, the C128 site of FBPase has been identified as a new allosteric site, where several nitrovinyl compounds can bind to inhibit FBPase activity. Herein, a series of nitrostyrene derivatives were further synthesized, and their inhibitory activities against FBPase were investigated in vitro. Most of the prepared nitrostyrene compounds exhibit potent FBPase inhibition (IC₅₀ < 10 μM). Specifically, when the substituents of F, Cl, OCH₃, CF₃, OH, COOH, or 2-nitrovinyl were installed at the R₂ (meta-) position of the benzene ring, the FBPase inhibitory activities of the resulting compounds increased 4.5–55 folds compared to those compounds with the same groups at the R₁ (para-) position. In addition, the preferred substituents at the R₃ position were Cl or Br, thus compound HS36 exhibited the most potent inhibitory activity (IC₅₀ = 0.15 μM). The molecular docking and site-directed mutation suggest that C128 and N125 are essential for the binding of HS36 and FBPase, which is consistent with the C128-N125-S123 allosteric inhibition mechanism. The reaction enthalpy calculations show that the order of the reactions of compounds with thiol groups at the R₃ position is Cl > H > CH₃. CoMSIA analysis is consistent with our proposed binding mode. The effect of compounds HS12 and HS36 on glucose production in primary mouse hepatocytes were further evaluated, showing that the inhibition was 71% and 41% at 100 μM, respectively.     

Reduced sedoheptulose-1,7-bisphosphatase levels in transgenic tobacco lead to decreased photosynthetic capacity and altered carbohydrate accumulation

Transgenic tobacco (Nicotiana tabacum L. cv. Samsun) plants with reduced levels of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) were produced using an antisense construct in which the expression of a tobacco SBPase cDNA clone was driven by the cauliflower mosaic virus (CaMV) promoter. The reduction in SBPase protein levels observed in the primary transformants correlated with the presence of the antisense construct and lower levels of the endogenous SBPase mRNA. No changes in the amounts of other Calvin cycle enzymes were detected using Western blot analysis. The SBPase antisense plants with less than 20% of wild-type SBPase activity were observed to display a range of phenotypes, including chlorosis and reduced growth rates. Measurements of photosynthesis, using both light-dosage response and CO₂ response curves, of T1 plants revealed a reduction in carbon assimilation rates, which was apparent in plants retaining 57% of wild-type SBPase activity. Reductions were also observed in the quantum efficiency of photosystem II. This decrease in photosynthetic capacity was reflected in a reduction in the carbohydrate content of leaves. Analysis of carbohydrate status in fully expanded source leaves showed a shift in carbon allocation away from starch, whilst sucrose levels were maintained in all but the most severely affected plants. Plants with less than 15% of wild-type SBPase activity were found to contain less than 5% of wild-type starch levels. The results of this preliminary analysis indicate that SBPase activity may limit the rate of carbon assimilation. Received: 23 February 1997 / Accepted: 2 May 1997     

Cloning,structure and expression of a pea cDNA clone coding for a photosynthetic fructose-1,6-bisphosphatase with some features different from those of the leaf chloroplast enzyme

A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in λgt11. The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative processing peptide. An additional 115 base pairs corresponding to a 3′-untranslated region coding for an mRNA poly(A)⁺ tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular mass 39253 daltons (Da), showing 82–88% absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase (kb)KpnI-SacI fragment of the λgt11 derivative was subcloned between theKpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates ofEscherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase. When assayed under excess Mg²⁺ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry. In contrast to the native pea chloroplast FBPase, theE. coli-expressed enzyme did not react with the monoclonal antibody GR-PB5. It also had a higher heat sensitivity, with 42% residual activity after heating for 30 min at 60°C, conditions which preserved the native enzyme in a fully active state. These results show the existence of some difference(s) in the conformation of the two FBPases; this could be a consequence of a different expression of the genomic and cDNA clones, or be due to the need for some factor for the correct assembly of the oligomeric structure of the native chloroplast enzyme. Accession number for pea chloroplast FBPase coding sequence: X68826 in the European Molecular Biology Laboratory (EMBL)     

Electrophoretic and chromatographic separation of two fructose-1,6-bisphosphatase forms from Synechococcus leopoliensix

d-Fructose-1,6-bisphosphatase (EC 3.1.3.11) activity in crude extracts of the blue-green alga Synechococcus leopoliensis (Anacystis nidulans) has been investigated using high resolving electrophoretic and chromatographic separation techniques. Two catalytically active enzyme forms which exhibited isoelectric points of 4.7–4.8 (designated from A) and 4.5–4.6 (designated form B) were resolved by isoelectric focusing. Both enzyme forms acted specifically on fluctose-1,6-bisphosphate. No interconversion between the A and B forms of fructose bisphosphatase activity was detected after refocusing. The apparent molecular weight of the two enzyme forms was determined by non denaturing polyacrylamide gradient electrophoresis; the values were 67,000–70,000 and 60,000–65,000 for A and B, respectively. Both enzyme forms were separated by preparative scale chromatofocusing. Kinetic measurements performed with the separated and partially purified fructose bisphosphatase forms indicated that both enzyme forms differ in their AMP sensitivity. The two enzymes were completely inactivated by the addition of cysteamine and reactivated by dithiols but the reactivation kinetics were different.Abbreviations DTT dl-Dithiothreithol - MTT 3(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide - PMS phenazine methosulfate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)-aminomethane     

Regulation of adenylate levels in intact spinach chloroplasts

Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts was determined and separated by electrophoresis in polyacrylamide gels. In spinach leaf extracts, a specific activity of 16 nmol glucose 1-phosphate formed per min per mg protein was found, whereas a lower value (6 nmol per min per mg protein) was observed in preparations of isolated chloroplasts which were about 75% intact. In the spinach leaf extracts two forms of phosphorylase were found; chloroplast preparations almost exclusively contained one of these. In pea leaf extracts the specific activity was 10 nmol glucose 1-phosphate formed per min per mg protein. Three forms of phosphorylase contributed to this activity. Preparations of isolated chloroplasts with an intactness of about 85% exhibited a lower specific activity (5nmol per min per mg protein) and contained two of these three phosphorylase forms.Abbreviations G1P Glucose 1-phosphate - P_i orthophosphate - Tris Tris (hydroxymethyl)aminomethane - MES 2(N-morpholino)ethane sulphonic acid - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

High level expression in Escherichia coli,purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves

In chloroplasts, the light-modulated fructose-1,6-bisphosphatase catalyzes the formation of fructose 6-bisphosphate for the photosynthetic assimilation of CO₂ and the biosynthesis of starch. We report here the construction of a plasmid for the production of chloroplast fructose-1,6-bisphosphatase in a bacterial system and the subsequent purification to homogeneity of the genetically engineered enzyme. To this end, a DNA sequence that coded for chloroplast fructose-1,6-bisphosphatase of rapeseed (Brassica napus) leaves was successively amplified by PCR, ligated into the Ndel/EcoRI restriction site of the expression vector pET22b, and introduced into Escherichia coli cells. When gene expression was induced by isopropyl--d-thiogalactopyranoside, supernatants of cell lysates were extremely active in the hydrolysis of fructose 1,6-bisphosphate. Partitioning bacterial soluble proteins by ammonium sulfate followed by anion exchange chromatography yielded 10 mg of homogeneous enzyme per 1 of culture. Congruent with a preparation devoid of contaminating proteins, the Edman degradation evinced an unique N-terminal amino acid sequence [A-V-A-A-D-A-T-A-E-T-K-P-]. Gel filtration experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the (recombinant) rapeseed chloroplast fructose-1,6-bisphosphatases was a tetramer [160 kDa] comprised of four identical subunits. Like other chloroplast fructose-1,6-bisphosphatases, the recombinant enzyme was inactive at 1 mM fructose 1,6-bisphosphate and 1 mM Mg²⁺ but became fully active after an incubation in the presence of either 10 mM dithiothreitol or 1 mM dithiothreitol and chloroplast thioredoxin. However, at variance with counterparts isolated from higher plant leaves, the low activity observed in absence of reductants was not greatly enhanced by high concentrations of fructose 1,6-bisphosphate (3 mM) and Mg²⁺ (10 mM). In the catalytic process, all chloroplast fructose-1,6-bisphosphatases had identical features; viz., the requirement of Mg²⁺ as cofactor and the inhibition by Ca²⁺. Thus, the procedure described here should prove useful for the structural and kinetic analysis of rapeseed chloroplast fructose-1,6-bisphosphatase in view that this enzyme was not isolated from leaves.Abbreviation DTT dithiothreitol - PCR polymerase chain reaction - EDTA (ethylenedinitrilo)tetraacetic     

Increased activity of the tandem fructose-1,6-bisphosphate aldolase,triosephosphate isomerase and fructose-1,6-bisphosphatase enzymes in <Emphasis Type="Italic">Anabaena</Emphasis> sp. strain PCC 7120 stimulates photosynthetic yield

The regulation of photosynthetic yield at the genetic level has largely focused on manipulation of the catalytic enzymes in the Calvin cycle by genetic engineering. In order to investigate the contribution of increased enzymatic activity in the Calvin cycle on photosynthetic yield, the rice fructose-1,6-bisphosphate aldolase (FBA), spinach triosephosphate isomerase (TPI) and wheat fructose-1,6-bisphosphatase (FBPase) genes were cloned in tandem and co-overexpressed in cyanobacterium Anabaena sp. strain PCC 7120 cells. The enzymatic activities of FBA, TPI and FBPase, as well as sedoheptulose-1,7-bisphosphatase (SBPase), were remarkably increased in transgenic cells relative to the wild-type. The photosynthetic yield, as reflected by photosynthetic O₂ evolution and dry cellular weight, was also markedly increased in transgenic cells versus wide-type cells. The activity of SBPase is considered the most important factor for ribulose-1,5-bisphosphate (RuBP) regeneration in the Calvin cycle, and increased activity of TPI alone in transgenic cells does not stimulate photosynthetic yield. Thus, the increased activity of FBA and FBPase, but not TPI, significantly improved photosynthetic yield in transgenic cells by stimulating SBPase activity and consequently accelerating the RuBP regeneration rate.     

In-vitro degradation of starch granules isolated from spinach chloroplasts

The initial reactions of transitory starch degradation in Spinacia oleracea L. were investigated using an in-vitro system composed of native chloroplast starch granules, purified chloroplast and non-chloroplast forms of phosphorylase (EC 2.4.1.1) from spinach leaves, and -amylase (EC 3.2.1.1) isolated from Bacillus subtilis. Starch degradation was followed by measuring the release of soluble glucans, by determining phosphorylase activity, and by an electron-microscopic evaluation following deep-etching of the starch granules. Starch granules were readily degraded by -amylase but were not a substrate for the chloroplast phosphorylase. Phosphorolysis and glucan synthesis by this enzyme form were strictly dependent upon a preceding amylolytic attack on the starch granules. In contrast, the non-chloroplast phosphorylase was capable of using starch-granule preparations as substrate. Hydrolytic degradation of the starch granules was initiated at the entire particle surface, independently of its size. As a result of amylolysis, soluble glucans were released with a low degree of polymerization. When assayed with these glucans as substrate, the chloroplast phosphorylase form exhibited a higher apparent affinity and a higher reaction velocity compared with the non-chloroplast phosphorylase form. It is proposed that transitory starch degradation in vivo is initiated by hydrolysis; phosphorolysis is most likely restricted to a pool of soluble glucan intermediates.Abbreviations Glc1P Glucose 1-phosphate - Mes 2(N-morpholino)ethanesulfonic acid - Pi Orthophosphate