Molecular and kinetic parameters of sugar transport across the frog choroid plexus

Summary The nature of sugar transport across the blood-cerebrospinal fluid barrier has been investigated using anin vitro preparation of the frog posterior choroid plexus. The permeability of 41 sugars and related compounds was measured by the rapid osmotic procedure described previously. Sugar permeation was found to be stereospecific, inhibited by 1,5-difluoro-2,4-dinitrobenzene, insensitive to anoxia, and independent of the external alkali cation composition. In addition, the transport of a sugar was inhibited by structural analogues. Transport occurred equally well from the ventricular or serosal surface of the tissue, and the rate of transport could be described formally by Michaelis-Menten kinetics. The results were analyzed in terms of the conformation of the sugars in aqueous solution. Sugars which were transported have the d-glucose chair conformation. There is a good correlation between the affinity of the sugar for the transport system and the number of hydroxyl groups attached to the equatorial plane of the ring; d-glucose with five equatorial hydroxyl groups has the greatest affinity. It is concluded that sugar transport across the choroid plexus occurs by facilitated diffusion.     

Properties of the sugar carrier in Baker's yeast

Incubation ofSaccharomyces cerevisiae cells withd-galactose induced the formation of galactose-utilizing enzymes, among them a monosaccharide carrier, apparently synthesized as a proteinde novo. The synthesis of the carrier preceded that of galactokinase by as much as 2 h. The inducible carrier shows a preference for monosaccharides with an axial hydroxyl group at carbon 4 of theC1 chair conformation or at carbon 2 of the1C chair conformation. Through its mediation, some sugars normally poorly transported (d-galactose,d-fucose,l-xylose,l-arabinose) can enter into the entire cell water, occupying then one more kinetic (and morphological ?) compartment than before induction. Some other monosaccharides, readily transported even by a constitutive carrier system (e.g.l-sorbose,d-xylose,d-arabinose) share the newly synthesized carrier.     

Suspension density and accumulation ratio of sugars and amino acids in yeasts

Suspension density has a pronounced effect on the transport parameters of monosaccharides, disaccharides and amino acids in all ycast species tested. InLodderomyces elongisporus, the accumulation ratio of 6-deoxy-D-glucose, a nonmetabolized sugar, was as high as 560: 1 at 0.5 mg dry mass per mL but only 10: 1 at 50 mg dry mass per mL. In the low-density range, the temperature optimum was very pronounced (at about 40 °C) and the pH optimum was very clear at pH 4.6. Iodoacetamide (0.5 mmol/L), 2,4-dinitrophenol (0.5 mmol/L), uranyl ions (0.5 mmol/L) and 2-deoxy-D-glucose (10 mmol/L) depressed the accumulation in the low-density range by 42, 97, 96 and 98 %, respectively. Preincubation with 1% sucrose and 1% L-fructose stimulated subsequent accumulation by 40 and 105%, respectively. In the high-density range, there was a poorly pronounced temperature optimum, no pH optimum and little effects of inhibitors except 2,4-dinitrophenol and 2-deoxy-D-glucose which inhibited by 68 and 89% respectively. No stimulation by preincubation with sugars was observed. There was a difference of 0.3 pH units in the intracellular pH of high-density and low-density cells and the membrane potential was -31 mV and -78 mV, respectively, which could not account for the differences in accumulation. However, there was a fine correlation between this accumulation ratio and the activity of the plasma membrane H⁺ATPase.     

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     

Transport properties of two extremely thermophilic species ofThermus

Thermus flavus and T. ruber grew optimally at 75 and 60 °C, respectively, but transport of monosaccharides (D-quinovose) and ammo acids (2-aminoisobutyric acid) had optima about 20 °C lower. Both transports were active, inhibited by 2,4-dinitrophenol but hardly at all by uranyl(2+) ions. Several transport systems are apparently involved with each class of compounds. Preincubation with glucose curtailed subsequent transport severely. Practical cessation of transport below 35 °C may be associated with the rather uniform composition of membrane lipids where iso- and anteiso-C₁₅ and C₁₇ acids are practically the only components.     

Densitometry of yeast cells and protoplasts during sugar uptake

Densitometry was applied to the study of sugar transport in yeast cells and protoplasts. The uptake curves of nonmetabolized monosaccharides were without the transient peak observed analytically. Differences in the space of distribution of different monosaccharides (e.g.d-arabinosev.l-arabinose) were confirmed. The uptake of metabolizable sugars (d-glucose,d-fructose) resulted in a gradual increase in protoplast (and cell) volume. The optical density of cells rose abruptly after some 20–60 min of incubation but this rise was not accompanied by a decrease in cell volume. The increase was prevented by 2,4-dinitrophenol but not by iodoacetic acid. The effect of temperature and of ionic strength of the medium was examined. Differences between protoplasts and intact cells in the utilization of sucrose were confirmed.     

Transport of histone intoEscherichia coli

Histone appears to be transported intoEscherichia coli cells in two ways: by an adsorption-like process and by a system which is temperature-dependent and is inhibited by 2,4-dinitrophenol. The adsorbed component cannot be washed out with a salt solution but can be partly exchanged for labelled histone and can be displaced with Tween 80. The actively transported histone can be replaced with unlabelled histone and with polylysine, and released by a high concentration of Mg²⁺ or by Tween 80. The active transport is observed only at concentrations greater than 100 μg histone/ml; the adsorption-like process occurring at lower concentrations.     

tight coupling of monosaccharide transport and metabolism inRhodotorula gracilis

A variety of agents have been applied to separate the uphill, metabolically linked transport of monosaccharides from facilitated diffusion inRhodotorula gracilis. The substances used were iodoacetamide, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, sodium azide, sodium fluoride and heavy-metal cations, including Cd²⁺, Hg²⁺, Cu²⁺, UO²⁺ ₂, La³⁺ and Th⁴⁺. No agent examined led to such separation. There was always either a complete inhibition of both cell metabolism (or uncoupling of oxidative phosphorylation) and the transport mechanism or the transport of sugars uphill persisted (when the metabolism was only partly inhibited). It is concluded that the monosaccharide transporting system inRhodotorula gracilis is tightly coupled to cell metabolism and cannot operate without sufficient energy supply even in cases when no uphill movement is involved.     

Transport ofl-tryptophan inSaccharomyces cerevisiae

In addition to the general amino acid transport system (GAP) ofS. cerevisiae l-tryptophan is transported by another system with approximately 25% capacity of GAP, with aK _T of 0.41±0.08 mmol/L and with a similar specificity as GAP (lower inhibition by Met, Pro, Ser, Thr and 2-aminoisobutyric acid; greater inhibition by Glu and His). The pH optimum of this system is at 5.0–5.5, activation energy above the transition point (20°C) was 20 kJ/mol, below the transition point 55 kJ/mol. The transport by this system was virtually unidirectional, efflux amounting to at most 10% into a tryptophan-free medium. The transport itself was blocked by 2,4-dinitrophenol, antimycin A and uranyl nitrate. The system was synthesized de novo during preincubation with glucose=fructose>trehalose >ethanol within 30 min, and was degraded with a half-time of 15 min in the absence of further synthesis. The accumulation ratios ofl-tryptophan ingap1 mutants were concentration-dependent (200∶1 at 1 μmoll-Trp/L, 4∶1 at 2.5 mmoll-Trp/L) and decreased with increasing suspension density from 200∶1 to 5∶1 (for 10 μmoll-Trp/L). The involvement of hydrogen ions in the uptake was clearly demonstrated by the effect of D₂O even if it could not be established by either shifts of pH_out or membrane depolarization.     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999     

Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides.

The virulence genes of Agrobacterium tumefaciens are induced by specific plant phenolic metabolites and sugars (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA, in press). In this report, monosaccharides, derivatives, and analogs which induce the vir regulon have been identified and the structural requirements for monosaccharide-mediated induction have been determined. Pyranose sugars with equatorial hydroxyls at C-1, C-2, and C-3 displayed strong vir gene-inducing activity; the C-4 hydroxyl could be epimeric and a wide variety of substitutions at C-5 were permissible. The acidic monosaccharide derivatives D-galacturonic acid and D-glucuronic acid were the strongest inducers among the monosaccharides tested. Eight of the 11 inducing compounds are known plant metabolites, and 7 are monomers of major plant cell wall polysaccharides. A role for monosaccharides and plant phenolic compounds as wound-specific plant metabolites which signal the ChvE/VirA/VirG regulatory system is proposed.     

Acceptor-specific glucuronyl transfer catalyzed by beta-glucuronidase.

Highly purified rat liver microsomal or lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) catalyzes the specific transfer of glucuronly residues from phenyl-beta-D-[U-14C]glucuronide to acceptor sugars. Specificity requirements of acceptor sugars are found to be: pyranose structure, 4C1-conformation and equatorial position of C2 and C3 hydroxyl groups or pyranose structure, 1C4-conformation and equatorial position of C3 and C4 hydroxyl groups. The acceptor capacities of 30 monosaccharides and glycosides including di- and tri- saccharides conform to this prinicple. The specificity of the beta-glucuronidase catalyzed glucuronyl transfer is proved by the exclusive formation of beta-glucuronly (1--3)glycosidic linkages. Glucuronly transfer rates increase with increasing donor substrate and increasing acceptor sugar concentration. In the presence of 1 M acceptor sugar the ratio of the transfer rate to the rate of enzymatic hydrolysis is about 2:1. An 'acceptor substrate binding site' on the surface of the beta-glucuronidase molecule which brings the C3 hydroxyl function of the acceptor sugar close enough to the C1 atom of the glucuronyl residue, is postulated.     

Carbohydrate Uptake and Metabolism of Ophiobolus graminis

The carbohydrate nutrition of Ophiobolus graminis, the cause of the take-all disease of wheat, was investigated in growth and respiration experiments. In a synthetic medium, d-mannitol was the only carbohydrate of thirteen studied which the fungus could not use for growth. However, the fungus was found to take up mannitol by an active mechanism, which was stopped by 2,4-dinitrophenol. Di- and trisaccharides were hydrolyzed extracellularly, and the monosaccharides were assimilated at different rates.     

The application ofin vivo techniques in the study of metabolic aspects of ion absorption in crop plants

Summary Little is known about the biochemical basis of the genotypic differences in the capacity for ion absorption and transport shown by many crop species. If these differences reflect the abundance of a specific membrane component or the activity of an enzyme we need to have some indication of thein vivo operation of these systems in whole plants. Thein vivo assessment of glycolytic enzymes is illustrated by the effects of mannose on the transport of phosphate in maize varieties. The application of high resolution³¹P-NMR to the study of intermediary metabolismin vivo is also helpful in following transport capacity. The five-fold rise in respiratory rate that occurs when freshly cut potato slices are maintained in aerated water for 24 hours is accompained by the turning on of a wide range of biochemical systems. Major increases in the capacity for absorption of phosphate from low concentrations (0.1 μM–10 μM) and in the phosphorylative ability of the tissue are seen, indicating the synthesis of a carrier involved in phosphate transport. These capacities differ markedly between individual tissues of the tuber,i. e. pith, parenchyma, cortex and buds and large differences have been observed between comparable tissue from different varieties. Varieties grown under similar conditions have been compared and shown to exhibit different kinetics with respect to the development of the low concentration absorption site and in their sensitivity to the effects of uncouplers such as 2,4-dinitrophenol.     

Transport of monosaccharides in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25 degrees was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S](i)) and extracellular ([S](o)) glucose concentrations was increased by 0.4mm-phlorrhizin and 0.3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S](i)/[S](o) fell below 1.0 only at [S](o) higher than 0.5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K(m) 1.16mm; V(max.) 4.5mumoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0.1mm-2,4-dinitrophenol, 0.4mm-phlorrhizin and by the absence of external Na(+). 6. The kinetic parameters of galactose entry into the cells were: K(m) 1.5mm; V(max) 10mumoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0 degrees , was inhibited by 0.4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na(+)-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na(+)-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

Biodegradation of the pesticide 4,6-dinitro-ortho-cresol by microorganisms in batch cultures and in fixed-bed column reactors

A mixed culture of microorganisms able to utilize 4,6-dinitro-ortho-cresol (DNOC) as the sole source of carbon, nitrogen and energy was isolated from soil contaminated with pesticides and from activated sludge. DNOC was decomposed aerobically in batch cultures as well as in fixed-bed column reactors. Between 65% and 84% of the substrate nitrogen was released as nitrate into the medium, and 61% of the carbon from uniformly ¹⁴C-labelled DNOC was recovered as ¹⁴CO₂. The mixed microbial culture also decomposed 4-nitrophenol and 2,4-dinitrophenol but not 2,3-dinitrophenol, 2,6-dinitrophenol, 2,4-dinitrotoluene, 2,4-dinitrobenzoic acid or 2-sec-butyl-4,6-dinitrophenol (Dinoseb). Maximal degradation rates for DNOC by the bacterial biofilm immobilized on glass beads in fixed-bed column reactors were 30 mmol day⁻¹ (l reactor volume)⁻¹, leaving an effluent concentration of less than 5 μg l⁻¹ DNOC in the outflowing medium. The apparent K _s value of the immobilized mixed culture for DNOC was 17 μM. Degradation was inhibited at DNOC concentrations above 30 μM and it ceased at 340 μM, possibly because of the uncoupling action of the nitroaromatic compound on the cellular energy-transducing mechanism. Received: 27 March 1997 / Received revision: 5 June 1997 / Accepted: 7 June 1997     

Transport of volatile fatty acids across the hindgut of the cockroach Panesthia cribrata Saussure and the termite, Mastotermes darwiniensis Froggatt

Acetate ion was actively transported from the lumen across the colon wall of the cockroach Panesthia cribrata. A maximal initial rate of transport of 81 μmol/h was obtained when the concentration of acetate was increased 400 times the physiological level to over 2 M. The transport system could not be saturated. Transport was not affected by 2,4-dinitrophenol or cyanide nor was it dependent on the maintenance of a sodium gradient. Propionate was transported to a lesser extent, but butyrate was transported at rates comparable to acetate. Acetate was also actively transported by the paunch and upper colon of Mastotermes darwiniensis but the transport system could not be saturated. A maximal initial rate of 3 μmol/h was obtained.     

Sugar uptake by intestinal basolateral membrane vesicles

A high yield of membrane vesicles was prepared from the basolateral surface of rat intestinal cells using an N2 cavitation bomb and density gradient centrifugation. The membranes were enriched 10-fold and were free of significatn contamination by brush border membranes and mitochondria. The rate of D-E114C]glucose and L-E13H]glucose uptake into the vesicle was measured using a rapid filtration technique. D-Glucose equilibrated within the vesicles with a half-time 1/25th that for L-glucose. The stereospecific uptake exhibited saturation kinetics with a Km of approx. 44 mM and a V of approx. 110 nmol . mg-1 min-1 at 10 degrees C. The activation energy for the process was 14 kcal . mol-1 below 15 degrees C and it approached 3 kcal . mol-1 above 22 degrees C. Carrier-mediated uptake was eliminated in the presence of 1 mM HgCl2 and 0.5 mM phloretin. The rate of transport was unaffected by the absence or presence of sodium concentration gradients. Competition studies demonstrated that all sugars with the D-glucose pyranose ring chair conformation shared the transport system, and that, with the possible exception of the -OH group at carbon No. 1, there were no specific requirements for an equatorial -OH group at any position in the pyranose ring. In the case of alpha-methyl-D-glucoside its inability to share the D-glucose transport system may be due to steric hindrance posed by the -OCH3 group rather than by a specific requirement for a free hydroxyl group at the position in the ring. It is concluded that sugars are transported across the basolateral membrane of the intestinal epithelium by a facilitated diffusion system reminiscent of that in human red blood cells.     

Auxin transport at cellular level: new insights supported by mathematical modelling

The molecular basis of cellular auxin transport is still not fully understood. Although a number of carriers have been identified and proved to be involved in auxin transport, their regulation and possible activity of as yet unknown transporters remain unclear. Nevertheless, using single-cell-based systems it is possible to track the course of auxin accumulation inside cells and to specify and quantify some auxin transport parameters. The synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) are generally considered to be suitable tools for auxin transport studies because they are transported specifically via either auxin influx or efflux carriers, respectively. Our results indicate that NAA can be metabolized rapidly in tobacco BY-2 cells. The predominant metabolite has been identified as NAA glucosyl ester and it is shown that all NAA metabolites were retained inside the cells. This implies that the transport efficiency of auxin efflux transporters is higher than previously assumed. By contrast, the metabolism of 2,4-D remained fairly weak. Moreover, using data on the accumulation of 2,4-D measured in the presence of auxin transport inhibitors, it is shown that 2,4-D is also transported by efflux carriers. These results suggest that 2,4-D is a promising tool for determining both auxin influx and efflux activities. Based on the accumulation data, a mathematical model of 2,4-D transport at a single-cell level is proposed. Optimization of the model provides estimates of crucial transport parameters and, together with its validation by successfully predicting the course of 2,4-D accumulation, it confirms the consistency of the present concept of cellular auxin transport.     

Investigation of transport-associated phosphorylation of sugar in yeast mutants (snf3) lacking high-affinity glucose transport and in a mutant (fdp1) showing deficient regulation of initial sugar metabolism

Pool-labeling experiments with 2-deoxyglucose in derepressed cells of the yeastSaccharomyces cerevisiae confirmed the previously reported results pointing to the possible existence of transport-associated phosphorylation of sugar. In yeast mutants containing a disruption or an inactivating point mutation in thesnf3 gene, which codes for the high-affinity glucose carrier, no evidence for transport-associated phosphorylation of 2-deoxyglucose was observed. If transport-associated phosphorylation in yeast exists, it is apparently not mediated by the low-affinity glucose carrier. Mediation by the high-affinity carrier would fit with the known requirement of an active kinase for high-affinity sugar transport. A mixed type of uptake in cells having both carriers would explain many of the problems associated with the 2-deoxyglucose pool-labeling experiments. Since mutants that have only low-affinity glucose transport are not deficient in the glucose-induced RAS-mediated cAMP signal, transport-associated phosphorylation of glucose is not required for or involved in the induction of the signal. The yeastfdp mutant, which dies on media containing fermentable sugars because of overaccumulation of sugar phosphates, also did not show any evidence for the existence of transport-associated phosphorylation. The same was true for the double mutantfdp snf3. The latter also showed the typicalfdp phenotype, indicative that the lethality on media containing fermentable sugar is owing to aberrant regulation of low-affinity transport. The high protein kinase activity in thefdp mutant does not appear to be responsible for the absence of evidence for transport-associated phosphorylation, because another mutant with high protein kinase activity, thebcy mutant, displayed normal transport behavior.