Substrate specifity of the hexose carrier in the plasmalemma of Chenopodium suspension cells probed by transmembrane exchange diffusion

Substrate specifity of the proton-driven hexose cotransport carrier in the plasmalemma of photoautotrophic suspension cells of Chenopodium rubrum L. has been studies through the short-term perturbation of ¹⁴C-labelled efflux of 3-O-methyl-d-glucose. Efflux, occurring exclusively via carrier-mediated exchange diffusion, is trans-stimulated by the substrate and trans-inhibited by the glucose-transport inhibitors phlorizin (K _1/2=7.9 mM) and its aglucon phloretin (K _1/2=84 μM); with both inhibitors, 3-O-methyl-d-glucose efflux may be blocked completely. Trans-stimulation of efflux (up to fourfold) by a variety of the d-enantiomers of neutral hexoses, including glucose (K _1/2=48 μM), 3-O-methyl-d-glucose (K _1/2=139 μM), and fructose (K _1/2=730 μM), but not by, for instance, d-allose, and l-sorbose, shows that carrier-substrate interaction critically involves the axial position at C-1 and C-3, respectively. We suggest that substrate binding by the Chenopodium hexose carrier involves both hydrophobic interaction with the pyran-ring and hydrogen-ion bonding at C-1 and C-3 of the d-glucose conformation.     

Carbohydrate and carbon metabolite accumulation responses in leaves of ozone tolerant and ozone susceptible spinach plants after acute ozone exposure

The objective of this study was to determine whether exposure of plants to ozone (O₃) increased the foliar levels of glucose, glucose sources, e.g., sucrose and starch, and glucose-6-phosphate (G6P), because in leaf cells, glucose is the precursor of the antioxidant, L-ascorbate, and glucose-6-phosphate is a source of NADPH needed to support antioxidant capacity. A further objective was to establish whether the response of increased levels of glucose, sucrose, starch and G6P in leaves could be correlated with a greater degree of plant tolerance to O₃. Four commercially available Spinacia oleracea varieties were screened for tolerance or susceptibility to detrimental effects of O₃ employing one 6.5 hour acute exposure to 25O nL O₃ L^-1 air during the light. One day after the termination of ozonation (29 d post emergence), leaves of the plants were monitored both for damage and for gas exchange characteristics. Cultivar Winter Bloomsdale (cv Winter) leaves were least damaged on a quantitative grading scale. The leaves of cv Nordic, the most susceptible, were approximately 2.5 times more damaged. Photosynthesis (Pn) rates in the ozonated mature leaves of cv Winter were 48.9% less, and in cv Nordic, 66.2% less than in comparable leaves of their non-ozonated controls. Stomatal conductance of leaves of ozonated plants was found not to be a factor in the lower Pn rates in the ozonated plants. At some time points in the light, leaves of ozonated cv Winter plants had significantly higher levels of glucose, sucrose, starch, G6P, G1P, pyruvate and malate than did leaves of ozonated cv Nordic plants. It was concluded that leaves of cv Winter displayed a higher tolerance to ozone mediated stress than those of cv Nordic, in part because they had higher levels of glucose and G6P that could be mobilized during diminished photosynthesis to generate antioxidants (e.g., ascorbate) and reductants (e.g., NADPH). Elevated levels of both pyruvate and malate in the leaves of ozonated cv Winter suggested an increased availability of respiratory substrates to support higher respiratory capacity needed for repair, growth, and maintenance.Abbreviations ADPG-PPiase ADPglucose pyrophosphorylase - ASC L-ascorbic acid - APX ascorbate peroxidase - Ce CO₂ concentration in air in the measuring cuvette during photosynthesis measurements - Ci CO₂ concentration in the leaf intercellular spaces during photosynthesis measurement - Chl chlorophyll - DHA dehydroascorbic acid - DHA reductase dehydroascorbate reductase - DHAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - Gluc glucose - GR glutathione reductase - G_sw stomatal conductance with units as mmol H₂O m^-2 s^-1 - GSSG oxidized glutathione - GSH reduced glutathione - G1P glucose-1-phosphate - G6P glucose-6-phosphate - G6P dehydrogenase glucose-6-phosphate dehydrogenase - 6PG 6-phosphogluconate - 6PG dehydrogenase 6-phosphogluconate dehydrogenase - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphate - MAL malate - MDHA reductase monodehydroascorbate reductase - PE post-emergence - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - PYR pyruvate - Pn net CO₂ photoas-similation in leaves - PPFD photosynthetic photon flux density with units of mol photons m^-2 s^-1 - PPRC pentose phosphate reductive cycle - RuBP ribulose-1,5-bisphosphate - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SLW specific leaf weight - TCA cycle tricarboxylic acid cycle - Triose-P DHAP+GAP     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

The effect of glyceraldehyde on red cells: Haemoglobin status,oxidative metabolism and glycolysis

Glyceraldehyde induces changes in the flux of glucose oxidised through the hexose monophosphate pathway, the concentrations of intermediates in the Embden-Meyerhoff pathway, the oxidative status of haemoglobin and levels of reduced and oxidised pyridine nucleotides and glutathione in red cells. Glyceraldehyde autoxidises in the cellular incubations, consuming oxygen and producing glyoxalase I- and II-reactive materials. Major fates of glyceraldehyde in red cells appear to be: (i) adduct formation with reduced glutathione and cellular protein; (ii) autoxidation and reaction with oxyhaemoglobin and pyridine nucleotides, and (iii) phosphorylation of d-glyceraldehyde and entry into the glycolytic pathway as glyceraldehyde 3-phosphate. The production of glycerol from glyceraldehyde by red cell l-hexonate dehydrogenase appears not to be a major reaction of glyceraldehyde in red cells. These results indicate that high concentrations of glyceraldehyde (1–50 mM) may induce oxidative stress in red cells by virtue of the spontaneous autoxidation of glyceraldehyde, forming hydrogen peroxide and α-ketoaldehydes (glyoxalase substrates). The implications of glyceraldehyde-induced oxidative stress for the in vitro anti-sickling effect of dl-glyceraldehyde and for the polyol pathway metabolism of glyceraldehyde are discussed.     

Transport and transporters in the endoplasmic reticulum

Enzyme activities localized in the luminal compartment of the endoplasmic reticulum are integrated into the cellular metabolism by transmembrane fluxes of their substrates, products and/or cofactors. Most compounds involved are bulky, polar or even charged; hence, they cannot be expected to diffuse through lipid bilayers. Accordingly, transport processes investigated so far have been found protein-mediated. The selective and often rate-limiting transport processes greatly influence the activity, kinetic features and substrate specificity of the corresponding luminal enzymes. Therefore, the phenomenological characterization of endoplasmic reticulum transport contributes largely to the understanding of the metabolic functions of this organelle. Attempts to identify the transporter proteins have only been successful in a few cases, but recent development in molecular biology promises a better progress in this field.     

Structural and serological characterization of the O-chain polysaccharide of Aeromonas salmonicida strains A449, 80204 and 80204-1

The O-chain polysaccharide (O-PS) of Aeromonas salmonicida was studied by a combination of compositional, methylation, CE-ESMS and one- and two-dimensional NMR analyses. It was found to be a branched polymer of trisaccharide-repeating units composed of L-rhamnose (Rha), D-glucose (Glc), 2-acetamido-2-deoxy-D-mannose (ManNAc) and O-acetyl group (OAc) and having the following structure: CE-ESMS analysis of A. salmonicida cells from strains A449, 80204 and 80204-1 grown under different conditions confirmed that the O-PS structure was conserved. ELISA-based serological study with native LPS-specific antisera performed on the native O-PS and its O-deacetylated and periodate-oxidized derivatives confirmed the importance of the O-PS backbone structure as an immunodominant determinant.     

Post-translational inhibitory regulation of acid invertase induced by fructose and glucose in developing apple fruit

Acid invertase (EC 3.2.1.26) is one of the key enzymes involved in the carbohydrate sink-organ development and the sink strength modulation in crops. The experiment conducted with 'Starkrimson' apple (Malus domestica Borkh) fruit showed that, during the fruit development, the activity of acid invertase gradually declined concomitantly with the progressive accumulation of fructose, glucose and sucrose, while Western blotting assay of acid invertase detected a 30 ku peptide of which the immuno-signal intensity increased during the fruit development. The im-muno-localization via immunogold electron microscopy showed that, on the one hand, acid invertase was mainly located on the flesh cell wall with numbers of the immunosignals present in the vacuole at the late stage of fruit development; and on the other hand, the amount of acid invertase increased during fruit development, which was consistent with the results of Western blotting. The in vivo pre-incubation of fruit discs with soluble sugars showed that