Effects of the anticancer agent VM-26 on hexose uptake in Ehrlich cells

The chemotherapeutic agent VM-26 is a membrane-interactive drug which we have previously demonstrated to be a potent inhibitor of nucleoside transport. Since the carriers mediating nucleoside and hexose transport are structurally and functionally similar, we have further characterized the membrane related properties of this agent by examining its effect on the transport and phosphorylation of hexoses in Ehrlich ascites cells. Under conditions in which only the transport component of hexose uptake was measured, VM-26 had no effect on the influx of 2-deoxyglucose, 3-0-methylglucose, or D-glucose. Glucose-sensitive cytochalasin B binding was only weakly inhibited by the drug. However, VM-26 was an apparent non-competitive inhibitor of the net uptake of 2-deoxyglucose (transport and phosphorylation). Measurement of hexokinase activity in cell extracts failed to demonstrate any significant effect of VM-26 on enzyme activity. In summary, although VM-26 is a potent inhibitor of the transport of nucleosides, it has no apparent effect on the transmembrane flux of hexoses indicating a differential effect on nucleoside and hexose transporters. The ability of the drug to decrease the net accumulation of hexoses in the absence of any detectable effect on hexokinase activity warrants further investigation.     

Different Sugar Kinases Are Involved in the Sugar Sensing of Galdieria sulphuraria

The unicellular acidophilic red alga Galdieria sulphuraria is a facultative heterotroph with a complex uptake system for sugars and polyols, consisting of at least 14 transporters. Upon transfer to heterotrophic conditions, these transporters were induced simultaneously. Once induced, transporters for common hexoses and pentoses are apparently not down-regulated under heterotrophic conditions. Uptake of deoxysugars (FUC/Rha), however, was repressed by substrates metabolized via gluco-, galacto-, glycero-, or hexokinase, whereas substrates phosphorylated by xylulokinase had no effect. This indicates that several sugar kinases play a key role in sugar sensing. In contrast, polyol transporters were repressed only by glucose or its analogs but not by other sugars. This repression does not involve the activity of kinases. Most likely this type of glucose sensing is independent of metabolism and takes place prior to or during uptake. In its natural environment, these two different sensing mechanisms would enable the alga to utilize a mixture of different substrates in a most economic way by repressing dispensible transporters.     

Distributed control of the glycolytic flux in wild-type cells and catabolite repression mutants of Saccharomyces cerevisiae growing in carbon-limited chemostat cultures

The sensitivity of the control of glycolysis was studied in the wild-type (WT) strain CEN.PK122 and in isogenic catabolite-repression mutants growing in carbon-limited, aerobic chemostat cultures at different dilution rates, D. Based on a model of glycolysis in which the glucose transport step was considered reversible and inhibited by glucose 6-phosphate (G6P), the matrix method of metabolic control analysis was applied. In the present work, we report that the control of glycolysis was significantly distributed between the glucose uptake, hexokinase, and phosphofructokinase steps. The flux control properties were sensitive to the glucose gradient through the membrane and the extent of inhibition of the transport by G6P as parameters of the glucose-uptake kinetics in all strains tested. In the WT strain at low and high D, most of the control was exerted by the phosphofructokinase (PFK)-catalyzed step. In the cat1 mutant, the step catalyzed by PFK was the most rate controlling while in the cat3 strain, the control was shared between the PFK, hexokinase (HK), and glucose transport steps. On the other hand, the mig1 mutant exhibited high control by the glucose transporter depending on the glucose gradient across the membrane. The results obtained are discussed in terms of the dependence upon the type of metabolism displayed by yeast and the kinetics of the sugar transport step.     

Characterisation of hexokinase in Toxoplasma gondii tachyzoites

We have cloned the hexokinase [E.C. 2.7.1.1] gene of Toxoplasma gondii tachyzoite and obtained an active recombinant enzyme with a calculated molecular mass of 51,465Da and an isoelectric point of 5.82. Southern blot analysis indicated that the hexokinase gene existed as a single copy in the tachyzoites of T. gondii. The sequence of T. gondii hexokinase exhibited the highest identity (44%) to that of Plasmodium falciparum hexokinase and lower identity of less than 35% to those of hexokinases from other organisms. The specific activity of the homogeneously purified recombinant enzyme was 4.04 micromol/mg protein/min at 37 degrees C under optimal conditions. The enzyme could use glucose, fructose, and mannose as substrates, though it preferred glucose. Adenosine triphosphate was exclusively the most effective phosphorus donor, and pyrophosphate did not serve as a substrate. K(m) values for glucose and adenosine triphosphate were 8.0+/-0.8 microM and 1.05+/-0.25mM, respectively. No allosteric effect of substrates was observed, and the products, glucose 6-phosphate and adenosine diphosphate, had no inhibitory effect on T. gondii hexokinase activity. Other phosphorylated hexoses, fructose 6-phosphate, trehalose 6-phosphate which is an inhibitor of yeast hexokinase, and pyrophosphate, also did not affect T. gondii hexokinase activity. Native hexokinase activity was recovered in both the cytosol and membrane fractions of the whole lysate of T. gondii tachyzoites. This result suggests that T. gondii hexokinase weakly associates with the membrane or particulate fraction of the tachyzoite cell.     

Transplacental carbohydrate and sugar alcohol concentrations and their uptakes in ovine pregnancy

The concentrations of glucose, fructose, sorbitol, glycerol, and myo-inositol in sheep blood and tissues have been reported previously (1--5). However, the other polyols that are at low concentrations have not been investigated in pregnant sheep due to technical difficulties. By using HPLC and gas chromatography-mass spectrometry, seven polyols (myo-inositol, glycerol, erythritol, arabitol, sorbitol, ribitol, and mannitol) and three hexoses (mannose, glucose, and fructose) were identified and quantified in four blood vessels supplying and draining the placenta (maternal artery, uterine vein, fetal artery, and umbilical vein). Uterine and umbilical blood flows were measured, and uptakes of all the polyols and hexoses in both maternal and fetal circulations were calculated. There was a significant net placental release of sorbitol to both maternal and fetal circulations. Fructose was also taken up significantly by the uterine circulation. Maternal plasma mannose concentrations were higher than fetal concentrations, and there was a net umbilical uptake of mannose, characteristics that are similar to those of glucose. Myo-inositol and erythritol had relatively high concentrations in fetal plasma (697.8 plus minus 53 microM and 463.8 plus minus 27 microM, respectively). The ratios of fetal/maternal plasma arterial concentrations were very high for most polyols. The concentrations of myo-inositol, glycerol, and sorbitol were also high in sheep placental tissue (2489 plus minus 125 microM/kg wet tissue, 2119 plus minus 193 microM/kg wet tissue, and 3910 plus minus 369 microM/kg wet tissue), an indication that these polyols could be made within the placenta.     

Hexose-specificity of hexokinase and ADP-dependence of pyruvate kinase play important roles in the control of monosaccharide utilization in freshly diluted boar spermatozoa

Incubation of boar sperm from fresh ejaculates in a minimal medium with 10 mM glucose induced a fast and intense activation of glycolysis, as indicated by the observed increases in the intracellular levels of glucose 6-phosphate (G 6-P) and ATP and the rate of formation of extracellular L-lactate. The effect of glucose was much more intense than that induced by fructose, sorbitol, and mannose. The greater utilization of glucose was related to a much greater sensitivity to hexokinase when compared with the other monosaccharides. Thus, the presence of 0.5 mM glucose induced total hexokinase activity in supernatants from sperm extracts of 1.7 +/- 0.1 mIU/mg protein, while the same concentration of both fructose, mannose, and sorbitol induced total hexokinase activity from 0.3 +/- 0.1 mIU/mg protein to 0.60 +/- 1 mIU/mg protein. Kinetic analysis of the total pyruvate kinase activity indicated that this activity was greatly dependent on the presence of ADP and also showed a great affinity for PEP, with an estimated Km in supernatants of 0.15-0.20 mM. Immunological location of proteins closely related to glycolysis, like GLUT-3 hexose transporter and hexokinase-I, indicated that these proteins showed the trend to be distributed around or in the cellular membranes of both head and midpiece in a grouped manner. We conclude that glycolysis is regulated by both the specific availability of a concrete sugar and the internal equilibrium between ATP and ADP levels. Furthermore, localization of proteins involved in the control of monosaccharide uptake and phosphorylation suggests that glycolysis starts at concrete points in the boar-sperm surface.     

Inhibition of the hexokinase/hexose transporter region in the glycosomal membrane of bloodstream Trypanosoma brucei by oligomycin and digitonin

Glycolysis in bloodstream T. brucei is the sole source of energy and remains a favourable chemotherapeutic target. In furtherance of this, an attempt has been made to understand better the contribution of glucose, fructose, mannose and glycerol to the energy charge of these parasites incubated in the presence of oligomycin, salicyhydroxamic acid (SHAM) and digitonin. Their cellular energy charge, when catabolizing glucose was 0.860, and under inhibition by oligomycin (10 microg), SHAM (2 mM) or oligomycin plus SHAM, 0.800, 0.444 and 0.405, respectively. Oligomycin inhibited the rate of catabolism of glucose, mannose and fructose up to 80%. The inhibition could not be alleviated by uncouplers, such as 2,4-dinitrophenol or permeabilization of the membranes by digitonin. Glucose-6-phosphate and other phosphorylated glycolytic intermediates, such as fructose-6-phosphate were catabolized by the permeabilized parasites in the presence of oligomycin, implying that except hexokinase, all the other glycolytic enzymes were active. Glucose oxidation was stimulated by low concentrations of digitonin (up to 4 microg), but at higher concentrations, it was significantly inhibited (up to 90% inhibition at 10 microg). Apparently, the inhibitory effects of oligomycin and digitonin were confined to glucose uptake and hexokinase catalysis. The above observations suggest that the hexose transporter and the enzyme hexokinase might be functionally-linked in the glycosomal membrane and oligomycin inhibits the linkage, by using a mechanism not linked to the energy charge of the cell. Digitonin at concentrations higher than 4 microg disrupted the membrane, rendering the complex in-operative. A hexokinase/hexose transporter complex in the glycosomal membrane is envisaged.     

Defective P2Y purinergic receptor function: A possible novel mechanism for impaired glucose transport

Extracellular ATP is an ubiquitous mediator that regulates several cellular functions via specific P2 plasma membrane receptors (P2Rs), for which a role in modulating intracellular glucose metabolism has been recently suggested. We have investigated glucose uptake in response to P2Rs stimulation in fibroblasts from type 2 diabetic (T2D) patients and control subjects. P2Rs expression was evaluated by RT-PCR; intracellular calcium release by fluorometry; glucose transporter (GLUT1) translocation by immunoblotting and chemiluminescence; glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose (2-DOG) and ATP by luminometry. Cells from T2D patients, in contrast to those from healthy controls, showed no increase in glucose uptake after ATP stimulation; extracellular ATP caused, however, a similar GLUT1 recruitment to the plasma membrane in both groups. P2Rs expression did not differ between fibroblasts from diabetic and healthy subjects, but while plasma membrane depolarization, a P2X-mediated response was similar in both groups, no evident intracellular calcium increase was detectable in the cells from the former group. The calcium response in fibroblasts from diabetics was restored by co-incubation with apyrase or hexokinase, suggesting that P2YRs in those cells were normally expressed but chronically desensitised. In support to this finding, fibroblasts from T2D subjects secreted a two-fold larger amount of ATP compared to controls. Pre-treatment with apyrase or hexokinase also restored ATP stimulated glucose uptake in fibroblasts from diabetic subjects. These results suggest that extracellular ATP plays a role in the modulation of glucose transport via GLUT1, and that the P2Y-dependent GLUT1 activation is deficient in fibroblasts from T2D individuals. Our observations may point to additional therapeutic targets for improving glucose utilization in diabetes.     

Potential role for the sorbitol pathway in the meiotic dysfunction exhibited by oocytes from diabetic mice

Complications common to type I diabetes, such as cataracts and cardiovascular disorders, have been associated with activation of the polyol pathway, which converts glucose to fructose via the intermediate, sorbitol. Under normal glycemic conditions, glucose is typically targeted for glycolysis or the pentose phosphate pathway through phosphorylation by hexokinase. When glucose levels are elevated under diabetic conditions, hexokinase becomes saturated, and the excess glucose is then shunted to aldose reductase, which converts glucose to sorbitol. In the present study, we examined the potential effects of this pathway on the maturation process in mouse oocytes. Increasing concentrations of sorbitol suppressed FSH-induced maturation in oocytes from control mice. Culturing oocytes from diabetic mice in the presence of inhibitors of aldose reductase reversed the suppression of FSH-induced meiotic maturation. When oocytes from control mice were cultured with activators of aldose reductase, FSH-induced maturation was compromised. In addition, treatment with sorbitol or activators of the polyol pathway led to reduced cell-cell communication between the oocyte and the cumulus cells, as well as compromised FSH-mediated cAMP production and de novo purine synthesis. These data indicate that the suppression of FSH-induced meiotic maturation observed in oocytes from diabetic mice may result from a shunting of glucose through the polyol pathway.     

Endothelin-1 stimulates the translocation and upregulation of both glucose transporter and hexokinase in astrocytes: relationship with gap junctional communication

We have previously shown that endothelin-1 increases glucose uptake in astrocytes. In the present work we investigate the mechanism through which endothelin-1 (ET-1) increases glucose uptake. Our results show that ET-1 activates a short-term and a long-term mechanism. Thus, ET-1 induced a rapid change in the localization of both GLUT-1 and type I hexokinase. These changes are probably aimed at rapidly increasing the entry and phosphorylation of glucose. In addition, ET-1 upregulated GLUT-1 and type I hexokinase and induced the expression of isoforms not normally expressed in astrocytes, such as GLUT-3 and type II hexokinase. These changes provide astrocytes with the machinery required to sustain a high rate of glucose uptake for a longer period of time. Our previous work had suggested that the effect of ET-1 on glucose uptake was associated with the inhibition of gap junctions. In this work, we compare the effect of ET-1 with that of carbenoxolone, a classical inhibitor of gap junction communication. Carbenoxolone increased glucose uptake to the same extent as ET-1 following the same mechanisms. Thus, carbenoxolone induced a rapid change in the localization of both GLUT-1 and type I hexokinase, upregulated GLUT-1 and type I hexokinase and induced the expression of GLUT-3 and type II hexokinase. When the inhibition of gap junction was prevented by tolbutamide, neither ET-1 nor carbenoxolone were able to increase the levels of GLUT-1, GLUT-3, type I hexokinase or type II hexokinase, indicating that these events are closely related to gap junctions.     

The interaction of phosphorylated sugars with human hexokinase I

Glucose 6-phosphate as well as several other hexose mono- and diphosphates were found by kinetic studies to be competitive inhibitors of human hexokinase I (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) versus MgATP. Limited proteolysis by trypsin does not destroy the hexokinase activity but produces as well-defined peptide map when the digested enzyme is electrophoresed in the presence of sodium dodecyl sulfate. MgATP at subsaturating concentration protects hexokinase from trypsin digestion, while phosphorylated sugars, Mg2+, glucose and inorganic phosphate have no effect. Addition of glucose 6-phosphate to the MgATP-hexokinase complex at a concentration 100-times higher than its Ki was not able to reverse the MgATP-induced conformation of hexokinase, suggesting that the binding of glucose 6-phosphate and MgATP are not mutually exclusive. Similar evidence was also obtained by studies of the induced modifications of ultraviolet spectra of hexokinase by the binding of MgATP, glucose 6-phosphate and both compounds. Among a library of monoclonal antibodies produced against rat brain hexokinase I and that recognize human placenta hexokinase I, one (4A6) was found to be able to modify the Ki of glucose 6-phosphate (from 25 to 140 microM) for human hexokinase I. The same antibody also weakens the inhibition by all the other hexoses phosphate studied without affecting the apparent Km for MgATP (from 0.6 to 0.75 mM) or for glucose. These data support the view for the binding of glucose 6-phosphate at a regulatory site on the enzyme.     

Competitive inhibition by glucose of myo-inositol incorporation into cultured porcine aortic endothelial cells

To explore the significance of hyperglycaemia as a causal factor for the appearance of diabetic angiopathies we investigated aspects of myo-inositol metabolism in porcine aortic endothelial cells. myo-Inositol was shown to be a long-living metabolite. Its uptake into the cells was mediated by a high-affinity, Na(+)-dependent uptake system inhibitable by ouabain with an apparent KM of 18.6 mumols/l, which was responsible for more than 80% of total uptake at physiological myo-inositol concentrations. Inhibition of inositol uptake by D-glucose was exclusively competitive with an apparent Ki of 24 mmol/l as shown by Lineweaver-Burk- and Dixon-plot analysis. The specificity of competitive inhibition was studied. L-Glucose which is stereochemically related to myo-inositol in the same way as the D-isomer proved to be an equally potent inhibitor. The hexoses D-galactose, D-mannose and D-fructose inhibited myo-inositol uptake to a minor extent. D-allose and 3-O-methyl-D-glucose had no inhibitory effect indicating that the OH-group of the carbon atom in 3 position is essential for the interaction with the carrier. The acyclic hexitol sorbitol also did not compete. As expected, the aldose reductase blocker sorbinil did not influence the carrier since there is no polyol pathway operating in porcine aortic endothelial cells. In accordance with the results of the uptake experiments, the incorporation of exogenous myo-inositol into membrane phosphatidylinositol was reduced at elevated extracellular glucose levels. The results raise the possibility that hyperglycaemia impairs endothelial inositol supply.     

Transport of hexoses by the phloem of Ricinus communis L. seedlings

The sieve-tube sap of Ricinus communis L. seedlings has been analysed to determine whether or not hexoses can be taken up by the phloem. Under natural conditions, i.e. with the endosperm attached to the cotyledons, glucose and fructose occurred only in trace amounts in the sieve-tube sap. Incubation of the cotyledons with hexoses in the concentration range 25–200 mM caused a rapid and substantial uptake of hexoses into the phleom, where they appeared eventually in the sieve-tube sap at the same concentration as in the incubation medium. Phloem loading of glucose, 3-O-methyl-glucose and sorbitol occurred easily, whereas fructose was less well loaded. glucose and to a larger extent fructose were also transformed to sucrose, which was loaded into the phloem. The loading of hexoses into the sieve tubes as observed in the experimental exudation system also occurred in the intact seedling, but transloction in the latter soon came to a standstill, probably because of lack of consumption by the sink tissues. These results indicate that the virtual absence of hexoses in the sievetube sap under in-vivo conditions is not because of the inability of the phloem-loading system to transport the monosaccharides but because of the absence of sufficiently high concentrations in the apoplast.     

Histological and immunohistochemical studies on flower induction in the olive tree (Olea europaea L.)

The aim of this research was to study flower bud differentiation processes in two oil olive cultivars from Tuscan germplasm (Leccino and Puntino). The effect of fruit-set was studied using 'ON' (with fruits) and 'OFF' (without fruits) shoots. Axillary buds were periodically collected at different phenological stages, from endocarp sclerification (July) until budbreak in the following spring. Thin sections were analysed using histology (apex size), histochemistry (RNA, starch and soluble carbohydrates) and cytokinin immunocytochemistry (zeatin localisation). The micromorphological observations and histochemical procedures did not allow us to distinguish axillary buds sampled from 'ON' and 'OFF' shoots. Cytokinin immunocytochemistry revealed early different localisation patterns between 'ON' and 'OFF' samples. Zeatin accumulated only in 'OFF' axillary bud meristems, particularly in July, when endocarp sclerification of fruits from the previous flowering is taking place. At this time, a strong RNA signal was also observed. Both these signals were correlated with floral evocation, and their coincidence with a phenological stage of development provided a useful tool to determine the time when axillary buds switch from the vegetative to the reproductive phase.     

Alternate models for shared carriers or a single maturing carrier in hexose uptake into rabbit jejunum in vitro

The uptake (tissue accumulation) of three hexoses into rabbit jejunum was measured in a flux chamber in conditions of effective stirring. Glucose uptake was inhibited by galactose or 3-O-methylglucose: 1-40 mM galactose caused a progressive decline in glucose uptake; 1-5 mM 3-O-methylglucose inhibited glucose uptake but higher concentrations of 3-O-methylglucose had no further effect. When 1-40 mM 3-O-methylglucose was added to glucose plus galactose there was a further decrease in the uptake of glucose; adding 1-40 mM galactose to glucose plus 3-O-methylglucose also produced a decrease in glucose uptake. Both glucose and 3-O-methylglucose inhibited uptake of galactose but the pattern of inhibition varied between the two sugars. The uptake of 3-O-methylglucose was also inhibited by glucose and by galactose, but the uptake of 3-O-methylglucose in the presence of either galactose or glucose was no further reduced by adding the third hexose. Graphical analysis and analysis by non-linear regression both showed that neither the single Michaelis-Menten function, nor the single Michaelis-Menten-plus-competitive-inhibition function was appropriate for any of these data. The results are consistent with the hypothesis that either there are multiple (at least three) intestinal carriers for hexoses; alternatively that there is a single carrier whose transport properties for the three hexoses change differentially during cell maturation and migration up the villus.     

Glucose metabolism in the rat small intestine: the effect of glucose analogues on hexokinase activity (Short Communication)

The effect of intubation of starved rats with solutions of glucose, 3-O-methylglucose, mannose, 2-deoxyglucose and sorbitol on the subcellular location of hexokinase in the mucosa of the small intestine was studied. Glucose, 3-O-methylglucose and mannose caused the soluble hexokinase activity to rise to a value similar to that found in fed animals, whereas sorbitol and 2-deoxyglucose caused smaller increases.     

Hexokinase in Conducting Tissues1

The localization and properties of hexokinase have been studiedin the conducting bundles of leaf petioles of sugar-beet. Ithas been shown that 12 to 20 per cent of hexokinase activityis found in the mitochondrial fraction and more than 10 percent in the microsomal fraction. The remaining hexokinase activityis concentrated in the soluble fraction of the cell. The propertiesof hexokinase associated with the structural elements and withthe soluble portion of the cell are different. The hexokinasein mitochondria and microsomes is non-specific; it phosphorylatesboth glucose and fructose but possesses a much greater affinityfor glucose. Consequently the phosphorylation of fructose inthese fractions is almost completely inhibited in the presenceof glucose, whereas fructose has only a relatively small suppressiveeffect on glucose phosphorylation. In the soluble fraction of the cells which corresponds to thecytoplasm, the phosphorylation of each of the hexoses occursindependently of the presence of the other sugar. The phosphorylationof fructose in this fraction is intensified in the presenceof K+ ions (activator of fructokinase). On this basis it issuggested that the soluble fraction contains specific fructo-kinasealong with non-specific hexokinase. The results are compared with those obtained earlier on thecompetition of these sugars during their transport into thecells of conducting bundles. Here glucose is a strong inhibitorof the fructose transport, while the latter only slightly affectsthe influx of glucose. This gives grounds for the belief thatnon-specific hexokinase, localized on the membranes, can takepart in the transport of hexoses into the cell.     

The 15 N-terminal amino acids of hexokinase II are not required for in vivo function: analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae

The function of the N-terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form). This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short form protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild-type protein dimerizes. Kinetic studies show that the enzymatic activities are very similar to wild-type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form of the enzyme is similar to the wild-type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N-terminal amino acids of hexokinase II are not required in vivo either for phosphorylation of hexoses or for glucose repression.     

How hexoses and inhibitors influence the malate transport system in Zygosaccharomyces bailii

When grown in fructose or glucose the cells of Zygosaccharomyces bailii were physiologically different. Only the glucose grown cells (glucose cells) possessed an additional transport system for glucose and malate. Experiments with transport mutants had lead to the assumption that malate and glucose were transported by one carrier, but further experiments proved the existence of two separate carrier systems. Glucose was taken up by carriers with high and low affinity. Malate was only transported by an uptake system and it was not liberated by starved malate-loaded cells, probably due to the low affinity of the intracellular anion to the carrier. The uptake of malate was inhibited by fructose, glucose, mannose, and 2-DOG but not by non metabolisable analogues of glucose. The interference of malate transport by glucose, mannose or 2-DOG was prevented by 2,4-dinitrophenol, probably by inhibiting the sugar phosphorylation by hexokinase. Preincubation of glucose-cells with metabolisable hexoses promoted the subsequent malate transport in a sugar free environment. Preincubation of glucose-cells with 2-DOG, but not with 2-DOG/2,4-DNP, decreased the subsequent malate transport. The existence of two separate transport systems for glucose and malate was demonstrated with specific inhibitors: malate transport was inhibited by sodium fluoride and glucose transport by uranylnitrate. A model has been discussed that might explain the interference of hexoses with malate uptake in Z. bailii.Abbreviations 2,4-DNP 2,4-dinitrophenol - 2-DOG 2-deoxyglucose - 6-DOG 6-deoxyglucose - pCMB para-hydroxymercuribenzoate     

Glucose sensing based on the intrinsic fluorescence of sol-gel immobilized yeast hexokinase

In this study, we investigated measurements of the intrinsic fluorescence of yeast hexokinase as an assay for glucose and immobilization of the enzyme in a silica sol-gel matrix as a potential in vivo glucose sensor for use in patients with diabetes. The intrinsic fluorescence of hexokinase in solution (excitation=295 nm, emission=330 nm) decreased by 23% at a saturating glucose concentration of 1 mM (Kd=0.3 mM), but serum abolished the glucose-related fluorescence response. When entrapped in tetramethylorthosilicate-derived sol gel, hexokinase retained activity, with a 25% maximal glucose-related decrease in intrinsic fluorescence, and the saturation point was increased to 50 mM glucose (Kd=12.5 mM). The glucose response range was increased further (to 120 mM, Kd=57 mM) by a covering membrane of poly(2-hydroxyethyl) methacrylate. Unlike free enzyme, the fluorescence responses to glucose with sol-gel immobilized hexokinase, with or without covering membrane, were similar for buffer and serum. We conclude that fluorescence monitoring of sol-gel entrapped yeast hexokinase is a suitable system for development as an in vivo glucose biosensor.