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A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds.

To analyze sugar transport processes during seed development of fava bean, we cloned cDNAs encoding one sucrose and one hexose transporter, designated VfSUT1 and VfSTP1, respectively. sugar uptake activity was confirmed after heterologous expression in yeast. Gene expression was studied in relation to seed development. Transcripts were detected in both vegetative and seed tissues. In the embryo, VfSUT1 and VfSTP1 mRNAs were detected only in epidermal cells, but in a different temporal and spatial pattern. VfSTP1 mRNA accumulates during the midcotyledon stage in epidermal cells covering the mitotically active parenchyma, whereas the VfSUT1 transcript was specific to outer epidermal cells showing transfer cell morphology and covering the storage parenchyma. Transfer cells developed at the contact area of the cotyledonary epidermis and the seed coat, starting first at the early cotyledon stage and subsequently spreading to the abaxial region at the late cotyledon stage. Feeding high concentrations of sugars suppressed both VfSUT1 expression and transfer cell differentiation in vitro, suggesting a control by carbohydrate availability.

Carbohydrate Metabolism↗

Surface sulphydryl groups and hexose monophosphate pathway activity in resting human polymorphonuclear leucocytes.

The role of surface -SH groups on the hexose monophosphate pathway activity in resting human polymorphonuclear leucocytes was studied. Endotoxin, taurocholic acid and concanvalin A stimulated [I-14C]glucose oxidation to a greater degree than [6-14C]glucose oxidation. The stimulation of glucose-I-C-oxidation by endotoxin, taurocholic acid and concanavalin A could be prevented by p-chloromercurybenzene sulphonic acid, an inhibitor of surface -SH groups. In contrast, polymorphonuclear leucocytes from four patients with chronic granulomatous disease failed to show stimulation of glucose-I-C-oxidation by endotoxin, taurocholic acid or concanavalin A.

4-Chloromercuribenzenesulfonate↗

Intraerythrocyte pH, pCO2 and the hexose monophosphate shunt.

We have documented a rise in pCO2 when erythrocytes are haemolysed by freeze-thaw using solid CO2 and methanol. This is partially corrected by preventing leakage of CO2 into the system. The remaining increment is partially explained by stimulation of the hexose monophosphate shunt when erythrocytes are haemolysed by this technique.

Carbon Dioxide↗

Comparison of computer simulations of the F-type and L-type non-oxidative hexose monophosphate shunts with 31P-NMR experimental data from human erythrocytes.

Mathematical modelling was used to predict the behaviour of the two most favoured schemes for the operation of the non-oxidative hexose monophosphate shunt (HMS), the F-type and the L-type pathways. The models simulate the time courses of sugar-phosphate concentrations when various substrates are metabolized via each pathway. A 31P-NMR technique, with which to observe time courses of concentrations of sugar phosphates in a human red cell lysate, was developed. The accuracy of each hypothesised scheme was then evaluated by comparing predicted with observed data. The results were more consistent with time courses of sugar-phosphate levels predicted by the F-type (classical) pathway than those predicted by the L-type model. However, the accumulation of sedoheptulose 1,7-bisphosphate when a haemolysate was incubated with ribose 5-phosphated showed that the F-type pathway is not a complete description of the system of reactions. Transaldolase was demonstrated to be essential for the normal metabolism of sugar phosphates by haemolysates. The effects of the heat-inactivation of transaldolase on the metabolism of sugar phosphates were accurately predicted by the F-type model. The relevance of attempting to describe the reaction of the non-oxidative HMS as a distinct 'pathway' or 'cycle' is discussed.

Computer Simulation↗

Characterization of hexose oxidase from the red seaweed Chondrus crispus.

Hexose oxidase from the red seaweed, Chondrus crispus was purified to homogeneity. The enzyme appeared to be encapsulated in particles obtained after mechanical disintegration of the fronds. Liberation of the enzyme in soluble form required either waiting for the spontaneous development of a suitable microbial flora in the suspension, or treatment with a mixture of proteases (pronase). As deduced from (SDS/)PAGE, the enzyme has a molecular mass of 87 kDa and probably consists of subunits of 36 kDa and 25 kDa. The low isoelectric point of 2.8 and the presence of 25% (by mass) sugars indicate that the enzyme is a strongly acidic glycoprotein. The absorption spectrum of isolated enzyme minus that of the substrate-reduced enzyme, and the EPR spectrum of the free radical observed in the reduced enzyme revealed the presence of a flavin. This cofactor is probably covalently bound since flavins were not released upon denaturation of the enzyme by heat or acid treatment. Taking free FAD as a reference compound, the enzyme contains 1 mol flavin/mol enzyme. EPR spectroscopy of the purified preparation showed the presence of Cu2+. However, since the amount was substoichiometric, substrate addition did not affect the signal, and the addition of chelator or Cu2+ did not affect the activity, the presence of this metal ion seems adventitious. It is concluded that the large discrepancies between the presently and the previously reported [Sullivan, J. D. & Ikawa, M. (1973) Biochim. Biophys. Acta 309, 11-22] characteristics of the enzyme probably originate from the characterization of a contaminating protein in the latter case.

Alcohol Oxidoreductases↗

Influence of mutations in hexose-transporter genes on glucose repression in Kluyveromyces lactis.

The variability of Kluyveromyces lactis strains in sensitivity to glucose is correlated with genetic differences in Kluyveromyces hexose transporter (KHT) genes. The glucose sensitive strain JA6 was shown to contain an additional gene, KHT2, not found in strains that are less sensitive. KHT2 is tandemly arranged with KHT1 which is identical to the low-affinity transporter gene RAG1, except for the C-terminus. Sequence analysis indicated that most of KHT2 had been lost by a recombination event between KHT1 and KHT2 generating the chimeric gene RAG1. Recombination between KHT1 and KHT2 was also found in mutants of JA6 selected as 2-deoxyglucose resistant colonies. These mutants, like kht1 kht2 double mutants were unable to grow on glucose when respiration was blocked (Rag- phenotype) and glucose repression was strongly reduced. kht1 or kht2 single mutants of JA6 were Rag+ but still an influence of the kht mutations on glucose repression was detectable. Repression was not affected in a Rag- mutant deleted for the phosphoglucose isomerase gene suggesting that the influence of transporter genes on repression is not caused by a reduction of the glycolytic flux. The data rather suggest that sensitivity to glucose repression is dependent on the rate of glucose uptake.

Amino Acid Sequence↗

Hexose transport in the apical and basolateral membranes of enterocytes in chickens adapted to high and low NaCl intakes.

1. The effect of a low-NaCl diet (LS diet) on the properties of hexose transport across the brush-border and basolateral membranes of enterocytes from jejunum, ileum and rectum of the chicken was investigated. 2. In the brush-border membrane, LS adaptation had no effect on Km for alpha-methyl-D-glucoside while Vmax values were significantly reduced in the ileum and in the rectum. All Scatchard plots of specific [3H]phlorizin binding give a straight line, consistent with a single population of binding sites. Phlorizin binding vs. alpha-methyl-D-glucoside maximal transport rates showed a linear correlation. 3. In the basolateral membrane, the LS diet did not modify the Km for D-glucose but reduced the Vmax in the ileum and in the rectum. Scatchard plots of [3H]cytochalasin B binding support the view that there is a single transport system in this membrane. There was a linear correlation between cytochalasin B binding and D-glucose Vmax values. 4. The response of the chicken intestine to LS intake consists of a dramatic reduction in the number of glucose transporters in both apical and basolateral membranes of the rectum, an intermediate response in the ileum and no significant effects in the jejunum.

3-O-Methylglucose↗

Hexose monophosphate shunt activity in erythrocytes related to cell age.

Erythrocytes were separated by age using a combination of density centrifugation and counterflow centrifugation and tested for basal activity of the hexose monophosphate shunt (HMP-shunt) as well as the methylene blue-stimulated maximal capacity by measuring CO2 production. No significant differences were found in basal HMP-shunt activity, but the maximal methylene blue-stimulated activity of old erythrocytes reached only half of the activity of the total cell population. The maximal HMP-shunt activity showed a significant correlation with hexokinase activity, but not with glucose-6-phosphate dehydrogenase activity in all but the youngest cells. The sensitivity to oxidative stress was tested by measuring the kinetics of pyruvate kinase isolated from erythrocytes incubated in presence and absence of methylene blue. Pyruvate kinase kinetics were affected more in the old cell population than in the total cell population: the K0.5 for phosphoenol-pyruvate increased four times in the unseparated cells and eight times in old cells.

Carbon Radioisotopes↗

Inhibition of hexose monophosphate shunt in young erythrocytes by pyrimidine nucleotides in hereditary pyrimidine 5' nucleotidase deficiency.

Recent reports have suggested that haemolytic anaemia in pyrimidine 5' nucleotidase (P5'N) deficiency might be due to impaired erythrocyte hexose monophosphate shunt (HMS). To investigate the relationship between pyrimidine accumulation, HMS impairment and shortened red-cell survival, we tested glucose 6-phosphate dehydrogenase (G-6PD), HMS, P5'N activities and the UV spectrum in whole red cells and in red cells of different age from 2 P5'N-deficient patients with different degrees of haemolytic anaemia. In whole red cells we found a reduction of both G-6PD and stimulated HMS activity in the presence of a variable amount of pyrimidine nucleotides (37.79 and 17.88 mumol/gHb respectively). A drastic inhibition of stimulated HMS activity was already present in the lightest red-cell fractions from patient 1, who presented a more severe haemolytic anaemia. The variable degree of pyrimidines found among red cell fractions, with a minor accumulation in the older red cells, supports the hypothesis that pyrimidine accumulation and HMS impairment occur in the younger erythrocytes of P5'N-deficient patients.

5'-Nucleotidase↗

Bilirubin inhibits hexose-monophosphate shunt activity of phagocytosing neutrophils.

Neutrophil hexose monophosphate (H.M.P.) shunt activity was measured by the conversion of 14C-1-glucose to 14CO2 during ingestion of polystyrene latex particles. Unconjugated bilrubin at the concentration of 1 X 10(5) M was found to markedly inhibit H.M.P. shunt activity. Since H.M.P. shunt activity of neutrophils is an important prerequisite of microbicidal function, it is suggested that jaundiced newborns may be more susceptible to bacterial infections.

Albumins↗

Evidence from temperature studies that the human erythrocyte hexose transporter has a transient memory of its dissociated ligands.

The inhibition constant of L-sorbose efflux (Ki(sorbose)) from human erythrocytes for inhibition by D-glucose increases from 5.15 +/- 0.89 to 12.24 +/- 1.9 mM on cooling from 50 degrees C to 30 degrees C; the Ki(sorbose) of D-mannose increases similarly on cooling. The activation energy Ea(sorbose) of net L-sorbose exit from human erythrocytes is 62.9 +/- 3.1 kJ mol-1; but in the co-presence of 5 mM D-glucose Ea(sorbose) is reduced to 41.7 +/- 1.6 kJ mol-1 (P < 0.005). These data are consistent with the view that when D-glucose binds to the hexose transporter it leads to an activated transporter state which remains transiently activated after glucose dissociates; if L-sorbose binds to the excited state it is more mobile than otherwise and consequently the apparent Ki(sorbose) of D-glucose is raised. Cooling prolongs the decay time of the activated state; hence the Ki(sorbose) of D-glucose rises as temperature is reduced.

Biological Transport↗

The placental transfer of hexoses and polyols in the guinea-pig, as shown by umbilical perfusion of the placenta.

1. Experiments involving polyol and sugar transfer across the guineapig placenta have been described.2. Regarding the former, sorbitol, dulcitol and mannitol are all transferred at approximately the same rate and more slowly than meso-inositol. Erythritol is not transferred.3. Concerning the hexose transfers, glucose and galactose are transferred at approximately the same rate and more rapidly than fructose.4. With regard to glucose transfer, there appears to be a mechanism with the following properties: (a) It tends towards saturation under certain conditions. (b) It favours saturation rather than diffusion kinetics for forward flows. (c) It exhibits competition with fructose and with galactose. Glucose/galactose competition has been used to demonstrate uphill transport by counterflow. (d) Competitive inhibition by phlorrhizin or phloretin could not be demonstrated.

Alcohols↗

Hexose-6-phosphate dehydrogenase found in human liver.

Starch-gel electrophoresis of extracts of human liver revealed the presence of a new hexose-6-phosphate dehydrogenase that was slower-moving at pH 8.6 than the sex-linked glucose-6-phosphate dehydrogenase. When the gel plate was stained, galactose-6-phos phate being used as a substrate, this enzyme band stained intensely, but the sex-linked glucose-6-phosphate dehydro genase failed to stain. This new human enzyme may well be homologous with the autosomally inherited glucose-6-phosphate dehydrogenase of the deer mouse (Peromyscus maniculatus), re ported by Shaw and Barto.

Adolescent↗

Stimulation of the hexose monophosphate shunt in human neutrophils by ascorbic acid: mechanism of action.

The addition of either ascorbic acid or dehydroascorbic acid to a suspension of polymorphonuclear leukocytes caused a dramatic increase in the resting hexose monophosphate shunt activity. A sequence of reactions involving dehydroascorbate, reduced glutathione, and reduced nicotinamide adenine dinucleotide phosphate is described to explain this stimulation. This sequence could provide an alternate method of producing H(2)O(2) and a bactericidal mechanism which is independent of myeloperoxidase.

Ascorbic Acid↗

A 25-kilodalton fraction from Mycobacterium tuberculosis that inhibits hexose monophosphate shunt activity, lysozyme release, and H2O2 production: reversal by gamma interferon.

This study examined the effects of a 25-kilodalton (kDa) glycolipoprotein derived from Mycobacterium tuberculosis on phagocyte functions associated with antimicrobial activity. The 25-kDa fraction inhibited the ability of both polymorphonuclear cells and cultured monocytes to release lysozyme and produce hydrogen peroxide. In addition, the glycolipoprotein was capable of reducing hexose monophosphate shunt activity and interfered with the ability of polymorphonuclear cells to reduce Nitro Blue Tetrazolium. Inhibition of these antimicrobial systems was optimal at a 50-micrograms/ml concentration of the 25-kDa fraction. Gamma interferon, but not alpha interferon, partially reversed the inhibitory effect of the mycobacterial component in all of the systems assessed. These studies indicate important mechanisms in the understanding of the pathogenesis of tuberculosis and suggest that gamma interferon may have a therapeutic role in mycobacterial diseases.

Blood Bactericidal Activity↗

Mechanism for regulating the distribution of glucose carbon between the Embden-Meyerhof and hexose-monophosphate pathways in Streptococcus faecalis.

Glucose-adapted Streptococcus faecalis produced little if any (14)CO(2) from glucose-1-(14)C, although high levels of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) were detected in cell-free extracts. Metabolism of glucose through the oxidative portion of the hexose-monophosphate pathway was shown to be regulated in this organism by the specific inhibitory interaction of the Embden-Meyerhof intermediate, fructose-1, 6-diphosphate (FDP), with 6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase activity was unaffected by FDP. The S. faecalis 6-phosphogluconate dehydrogenase was partially purified from crude extracts by standard fractionation procedures and certain kinetic parameters of the FDP-mediated inhibition were investigated. The negative effector was shown to cause a decrease in V(max) and an increase in the apparent K(m) for both 6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP). These effects were apparently a consequence of the ligand interacting with the enzyme at a site distinct from either the substrate or the coenzyme sites. Among the evidence supporting this was the fact that beta-mercaptoethanol blocked completely FDP inhibition, but had no effect on catalytic activity. The possibility that the regulation of 6-phosphogluconate dehydrogenase activity by FDP might be of some general significance was suggested by the observation that this enzyme from several other sources was also sensitive to FDP.

Animals↗

Genetic Control of the Transport of Hexose Phosphates in Escherichia coli: Mapping of the uhp Locus.

A number of mutations affecting the transport of hexose phosphates in Escherichia coli were ordered within the uhp locus. Three-point crosses by transduction or conjugation allowed the ordering of the alleles relative to the adjacent pyrE marker. The same linear map was obtained by both methods. This, combined with the regulatory properties of revertants of these mutants, allowed a tentative identification of two genes, one presumably coding for the transport system (uhpT) and the other(s) specifying a regulatory element (uhpR). The order of these is pyrE-uhpT-uhpR. Mutants exhibiting constitutive expression of the transport system were isolated. This behavior is genetically linked to the uhp locus, but more precise localization was not possible.

Journal Article↗