Structural requirements for active intestinal transport. The nature of the carrier–sugar bonding at C-2 and the ring oxygen of the sugar

Several weakly transported sugars were tested for transport by the Na⁺-dependent sugar carrier with slices of everted hamster intestinal tissue. Sugars were assumed to be transported by this carrier if the accumulation was diminished in the absence of Na⁺ and in the presence of the competitive inhibitor 1,5-anhydro-d-glucitol. The extent of accumulation was correlated with the number of hydroxyl groups in the d-gluco configuration if the ring oxygen was placed in the normal d-glucose position. 5-Thio-d-glucose, with a sulphur atom in the ring, was transported at about the same rate as d-glucose and had a similar K_i for d-galactose transport, but myoinositol was poorly accumulated. It is suggested that there is no hydrogen bonding at the ring oxygen atom, but that the oxygen atom is found at this position as a result of steric constraints. No sugar without a hydroxyl group in the d-gluco position at C-2 of the sugar, including d-mannose, 2-deoxy-d-glucose, 2-chloro-2-deoxy-d-glucose and 2-deoxy-2-fluoro-d-glucose, was transported by the Na⁺-dependent carrier, but these sugars and l-fucose weakly and competitively inhibit the Na⁺-dependent accumulation of l-glucose into slices of everted hamster intestinal tissue. It is concluded that the bond between the carrier and C-2 of the sugar may be covalent, and a possible mechanism for active intestinal transport is proposed.     

Molecular cloning and characterization of two novel fructose-specific transporters from the osmotolerant and fructophilic yeast Candida magnoliae JH110

Sugar transport is very critical in developing an efficient and rapid conversion process of a mixture of sugars by engineered microorganisms. By using expressed sequence tag data generated for the fructophilic yeast Candida magnoliae JH110, we identified two fructose-specific transporters, CmFSY1 and CmFFZ1, which show high homology with known fructose transporters of other yeasts. The CmFSY1 and CmFFZ1 genes harbor no introns and encode proteins of 574 and 582 amino acids, respectively. Heterologous expression of the two fructose-specific transporter genes in a Saccharomyces cerevisiae, which is unable to utilize hexoses, revealed that both transporters are functionally expressed and specifically transport fructose. These results were further corroborated by kinetic analysis of the fructose transport that showed that CmFsy1p is a high-affinity fructose–proton symporter with low capacity (K _M?=?0.13?±?0.01 mM, V _max?=?2.1?±?0.3 mmol h^?1 [gdw]^?1) and that CmFfz1p is a low-affinity fructose-specific facilitator with high capacity (K _M?=?105?±?12 mM, V _max?=?8.6?±?0.7 mmol h^?1 [gdw]^?1). These fructose-specific transporters can be used for improving fructose transport in engineered microorganisms for the production of biofuels and chemicals from fructose-containing feedstock.     

Sugar Transport in Immature Internodal Tissue of Sugarcane: II. Mechanism of Sucrose Transport

The mechanism by which sucrose is transported into the inner spaces of immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L. var. H 49-5) was studied in short term experiments (15 to 300 seconds). Transport of sucrose, glucose, and fructose was each characterized by a V_max of 1.3 μmoles/gram fresh weight·2 hours, and each of these three sugars mutually and competitively inhibited transport of the other two. When ¹⁴C-glucose was supplied exogenously, ¹⁴C-glucose 6-phosphate and ¹⁴C-glucose were the first labeled compounds to appear in the tissue; no ¹⁴C-sucrose was detected until after 60-second incubation. After 15-second incubation in ¹⁴C-sucrose, all intracellular radioactivity was in glucose, fructose, glucose 6-phosphate, and fructose 6-phosphate; trace amounts of ¹⁴C-sucrose were found after 30 seconds and after 5 minutes, 71% of the intracellular radioactivity was in sucrose. Although it was possible that sucrose was transported intact into the inner space and then immediately hydrolyzed, it was shown that the rate of hydrolysis under these conditions was too low to account for the rate of hexose accumulation. Pretreatment of the tissue with rabbit anti-invertase antiserum eliminated sucrose transport, but had no effect on glucose transport. Since the antibodies did not penetrate the plasmalemma, it was concluded that sucrose was hydrolyzed by an invertase in the free space prior to transport. The glucose and fructose moieties, or their phosphorylated derivatives, were then transported into the inner space and sucrose was resynthesized. No evidence for the involvement of sucrose phosphate in transport was found in these experiments.     

In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm

Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net ¹⁴C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of ¹⁴C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q₁₀ suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis).     

Carbon stable isotopic composition of soluble sugars in Tillandsia epiphytes varies in response to shifts in habitat

We studied C stable isotopic composition (δ¹³C) of bulk leaf tissue and extracted sugars of four epiphytic Tillandsia species to investigate flexibility in the use of crassulacean acid metabolism (CAM) and C₃ photosynthetic pathways. Plants growing in two seasonally dry tropical forest reserves in Mexico that differ in annual precipitation were measured during wet and dry seasons, and among secondary, mature, and wetland forest types within each site. Dry season sugars were more enriched in ¹³C than wet season sugars, but there was no seasonal difference in bulk tissues. Bulk tissue δ¹³C differed by species and by forest type, with values from open-canopied wetlands more enriched in ¹³C than mature or secondary forest types. The shifts within forest habitat were related to temporal and spatial changes in vapor pressure deficits (VPD). Modeling results estimate a possible 4% increase in the proportional contribution of the C₃ pathway during the wet season, emphasizing that any seasonal or habitat-mediated variation in photosynthetic pathway appears to be quite moderate and within the range of isotopic effects caused by variation in stomatal conductance during assimilation through the C₃ pathway and environmental variation in VPD. C isotopic analysis of sugars together with bulk leaf tissue offers a useful approach for incorporating short- and long-term measurements of C isotope discrimination during photosynthesis.     

The accumulation and metabolism of [C]hypoxanthine by slices of rabbit renal medulla

The uptake of hypoxanthine by rabbit renal medulla has been studied with in vitro conditions. Unlike the uptake by renal cortex slices reported earlier, no evidence was found for involvement of an organic cation transport system. Medullary accumulation of the ¹⁴C-labeled material occurred in the absence of O₂ if glucose was present as substrate. Uptake of the ¹⁴C label was not supported by other sugars or metabolic intermediates, however. Metabolic inhibitors reduced both aerobic and anaerobic uptake. Tissue extracts were subjected to high-voltage electrophoresis and gel filtration for identification of possible metabolites. These studies indicated that most of the uptake of ¹⁴C-labeled material was accounted for by its conversion to inosine-like and/or inosinic acid-like compounds. That is, when experimental conditions were designed to retard slice metabolism of hypoxanthine, tissue to medium ratios for ¹⁴C approximated 1.0. A third metabolite was found occasionally, but remains unidentified.     

Development and other characteristics of α-methyl-d-glucoside transport by rat kidney cortex slices

α-Methyl-d-glucoside has been shown to be a non-metabolizable sugar which is accumulated against a concentration gradient by a Na⁺-dependent and phlorizin inhibited process by adult rat renal cortical slices incubatedin vitro at 37 °C. (2) The velocity of accumulation increased linearly with substrate concentrations up to 1.5 mM, but at higher concentrations obeyed saturable kinetics with an apparentK_m of about 6 mM. (3) Uptake was enhanced as Na⁺ was increased from 0 to 100 mequiv/l. Higher Na⁺ concentrations caused no further effect. (4) A pH maximum of transport occurred between 7.35 and 8.0. (5) Glucoside uptake was inhibited byd-glucose,d-galactose,d-fructose,d-mannose andd-ribose. The inhibition byd-glucose andd-galactose was competitive with apparentK_t of 24 and 53 mM, respectively. (6) Bothd-glucose andd-galactose accelerated the efflux of α-methyl-d-glucoside from preloaded cells. (7) Kidney cortex slices from 1-day-old rats were unable to accumulate α-methyl-d-glucoside to form a concentration gradient. The ability to concentrate the glucoside increased progressively after birth, reaching near normal in tissue from 15-day-old animals. The data indicate that the transport process in the newborn is rudimentary, failing also to display accelerated efflux phenomenon. (8) α-Methyl-d-glucoside is transported in rat kidney cortex by a mechanism similar in many ways to that ofd-galactose.     

The effect on amino acid transport of trypsin treatment of rat renal brush border membranes

Trypsin treatment of isolated rat renal brush border membrane vesicles which preferentially releases l-leucine aminopeptides (EC 3.4.11.2) decreases their ability to take up a variety of amino acids under Na⁺-gradient conditions. Such treatment did not alter the osmotic properties of the vesicles nor affect their fragility. A linear correlation could be demonstrated between the l-leucine aminopeptidase activity of the membranes and the initial rate of uptake of l-leucine and l-proline. Velocity of uptake-concentration dependence studies with these substrates indicate that the major effect of trypsinization is to decrease the maximum velocity (V_max1) of the low-K_m high-affinity system with little effect on the V_max2 of the high-K_m low-affinity transport process and no effect on the apparent Michaelis constants of either. Although the data indicate that l-leucine aminopeptidase activity and uptake of l-leucine and l-proline are affected in parallel, they should not be construed to imply a role of the enzyme in the transport process, especially in view of the global decrease in the uptake of various amino acids and sugars.     

Two components of auxin transport

The transport of indoleacetic acid-1-¹⁴C out of sunflower stem sections has been analyzed by a compartmental analysis procedure in which the radioactivity moving out of the tissue (log per cent) is plotted against time. The analysis indicates that indoleacetic acid is transported via a fast transport system (t_½ of about 30 minutes) and a slow transport system (t_½ about 10 hours). While we do not know the sources of these two pools, by analogy with ion transport studies, the fast efflux is characteristic of transport from the cytoplasm across the plasmalemma and the slow efflux is characteristic of transport across the tonoplast and thus out of the vacuole. Both components of transport are inhibited by 2,3,5-triiodobenzoic acid.     

Myo-inositol transport in the small intestine of the domestic fowl

• 1.1. Myo-inositol is transported in chicken small intestine by a mediated route with an apparent K_m of 0.1 mM and by a diffusion mechanism.
• 2.2. The mediated route is susceptible to inhibition by sugars, though sugars are not transported by this process, nor is myo-inositol transported by the sugar transport system.
• 3.3. Myo-inositol influx is inhibitable by phlorizin, sulfhydryl reagents, removal of Na⁺ from the incubation medium, and preloaded sugar; it can be stimulated by theophylline.
• 4.4. The ability to absorb this nutrient varies greatly between individual animals.

     

In vitro and in vivo modification of sugar transport and translocation in celery by phytohormones

In an attempt to address the controversy in the literature as to whether phytohormones have any direct effect on phloem loading of sucrose, we investigated the effect of gibberellic acid (GA₃) and indoleacetic acid (IAA) on sugar transport and translocation in celery (Apium graveolens L. cv. Utah 5270). Both hormones enhanced sucrose uptake into isolated vascular bundles and phloem tissue of celery and enhanced the export of ¹⁴C assimilates from leaves of intact plants in vivo. The hormone-induced increase of uptake into isolated vascular bundles or phloem was specific for sucrose and mannitol which are translocated in phloem. Furthermore, the hormone-induced increase in translocation was not due to an increase in sink demand, since neither glucose nor sucrose uptake rates were affected in the storage parenchyma tissue discs (sink region) in the presence of GA₃ or IAA. The evidence suggests that phytohormones may have a direct effect on phloem loading of sucrose. The possibility of short-term GA₃ and IAA effects on processes resulting in membrane transport of sugars in celery is discussed.     

Tolerance of combined submergence and salinity in the halophytic stem-succulent Tecticornia pergranulata

Background and Aims

Habitats occupied by many halophytes are not only saline, but are also prone to flooding. Few studies have evaluated submergence tolerance in halophytes.

Methods

Responses to submergence, at a range of salinity levels, were studied for the halophytic stem-succulent Tecticornia pergranulata subsp. pergranulata (syn. Halosarcia pergranulata subsp. pergranulata). Growth and total sugars in succulent stems were assessed as a function of time after submergence. Underwater net photosynthesis, dark respiration, total sugars, glycinebetaine, Na⁺, Cl⁻ and K⁺, in succulent stems, were assessed in a NaCl dose-response experiment.

Key Results

Submerged plants ceased to grow, and tissue sugars declined. Photosynthesis by succulent stems was reduced markedly when underwater, as compared with in air. Capacity for underwater net photosynthesis (P_N) was not affected by 10–400 mm NaCl, but it was reduced by 30 % at 800 mm. Dark respiration, underwater, increased in succulent stems at 200–800 mm NaCl, as compared with those at 10 mm NaCl. On an ethanol-insoluble dry mass basis, K⁺ concentration in succulent stems of submerged plants was equal to that in drained controls, across all NaCl treatments. Na⁺ and Cl⁻ concentrations, however, were elevated in stems of submerged plants, but so was glycinebetaine. Submerged stems increased in succulence, so solutes would have been ‘diluted’ on a tissue-water basis.

Conclusions

Tecticornia pergranulata tolerates complete submergence, even in waters of high salinity. A ‘quiescence response’, i.e. no shoot growth, would conserve carbohydrates, but tissue sugars still declined with time. A low K⁺ : Na⁺ ratio, typical for tissues of succulent halophytes, was tolerated even during prolonged submergence, as evidenced by maintenance of underwater P_N at up to 400 mm NaCl. Underwater P_N provides O₂ and sugars, and thus should enhance survival of submerged plants.Key words: Flooding, halophyte, Halosarcia pergranulata, inundation, inland salt marsh, respiration, Salicornioideae, salt lake, submergence–salinity interaction, tissue solutes, underwater net photosynthesis     

Photosynthesis in c(4) plant tissue cultures: significance of kranz anatomy to c(4) Acid metabolism in c(4) plants

The pattern of photosynthetic carbon metabolism was determined in tissue cultures of Portulaca oleracea. Four-carbon acids are the most heavily labeled photosynthetic products during short term exposure to ¹⁴CO₂, containing greater than 40% of the total radioactivity incorporated. Phosphoglyceric acid and sugars account for only 10% of the label after equal exposure times. Other features of the CO₂ assimilation pattern in Portulaca callus tissue include a relatively large percentage of label located in various minor products throughout the time course studied, and a greater incorporation of ¹⁴C into sugars in tissue cultures than occurs in leaves. Ultrastructurally, the chloroplasts and cells of the callus are like those in the mesophyll cells of Portulaca leaves. The requirement for Kranz anatomy for operation of functional C₄ physiology is discussed.     

Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles

Pea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K_m of about 0.23 millimolar for MgATP, is only slightly affected by K⁺ or Cl⁻ and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO₃⁻ > Cl⁻) and has a K_m of about 0.7 millimolar. Auxins decrease the K_m to about 0.35 millimolar, with no significant effect on the V_max, while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport.     

Transport of 3-O-methyl d-glucose and β-methyl d-glucoside by rabbit ileum

The intestinal transport of three actively transported sugars has been studied in order to determine mechanistic features that, (a) can be attributed to stereospecific affinity and (b) are common.The apparent affinity constants at the brush-border indicate that sugars are selected in the order, $\text{β-methyl glucose >}\text{d}\text{-galactose}\text{> 3-O-}\text{methyl}$ glucose, (the K_m values are 1.23, 5.0 and 18.1 mM, respectively.) At low substrate concentrations the K_t values for Na⁺ activation of sugar entry across the brush-border are: 27.25, and 140 mequiv. for β-methyl glucose, galactose and 3-O-methyl glucose, respectively. These kinetic parameters suggest that Na⁺, water, sugar and membrane-binding groups are all factors which determine selective affinity.In spite of these differences in operational affinity, all three sugars show a reciprocal change in brush-border entry and exit permeability as Ringer [Na] or [sugar] is increased. Estimates of the changes in convective velocity and in the diffusive velocity when the sugar concentration in the Ringer is raised reveal that with all three sugars, the fractional reduction in convective velocity is approximately equal to the (reduction of diffusive velocity)². This is consistent with the view that the sugars move via pores in the brush-border by convective diffusion.Theophylline reduces the serosal border permeability to β-methyl glucose and to 3-O-methyl glucose relatively by the same extent and consequently, increases the intracellular accumulation of these sugars.The permeability of the serosal border to β-methyl glucose entry is lower than permeability of the serosal border to β-methyl glucose exit, which suggests that β-methyl glucose may be convected out of the cell across the lateral serosal border.     

Models of prebiological phosphorylation

The hypothesis that contemporary metabolic pathways evolved from analogous chemical reaction sequences on the primitive Earth leads to a reexamination of models of prebiological phosphorylation. Present-day phosphate uptake by algae and bacteria seems to involve two transport systems: (a) An active transport process occurring at low external phosphate concentrations (as in unpollusive) process at higher phosphate concentrations (>10⁻⁶ M) (as in the interstitial water of reducing sediments). Laboratory model experiments are described for the reaction of reducing sugars with orthophosphate in the presence of cyanogen, producing glycosyl phosphates. These reactions proceed with appreciable yields only at high phosphate concentrations (>10⁻³ M), and may thus possibly serve as simulations of prebiological phosphorylation with diffusive transport, as it may have occurred in the interstitial water of reducing sediments.     

Intracellular pH Regulation during NO(3) Assimilation in Shoot and Roots of Ricinus communis

Ricinus communis L. was used to test the Dijkshoorn-Ben Zioni hypothesis that NO₃⁻ uptake by roots is regulated by NO₃⁻ assimilation in the shoot. The fate of the electronegative charge arising from total assimilated NO₃⁻ (and SO₄²⁻) was followed in its distribution between organic anion accumulation and HCO₃⁻ excretion into the nutrient solution. In plants adequately supplied with NO₃⁻, HCO₃⁻ excretion accounted for about 47% of the anion charge, reflecting an excess nutrient anion over cation uptake. In vivo nitrate reductase assays revealed that the roots represented the site of about 44% of the total NO₃⁻ reduction in the plants. To trace vascular transport of ionic and nitrogenous constituents within the plant, the composition of both xylem and phloem saps was thoroughly investigated. Detailed dry tissue and sap analyses revealed that only between 19 and 24% of the HCO₃⁻ excretion could be accounted for from oxidative decarboxylation of shoot-borne organic anions produced in the NO₃⁻ reduction process. The results obtained in this investigation may be interpreted as providing direct evidence for a minor importance of phloem transport of cation-organate for the regulation of intracellular pH and electroneutrality, thus practically eliminating the necessity for the Dijkshoorn-Ben Zioni recycling process.     

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane–based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine K_M, V_max, K_D, and k_obs values for different sugar substrates. K_D and k_obs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H⁺ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H⁺ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s⁻¹) and for sugar translocation (2 s⁻¹ − 30 s⁻¹ for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.     

Photosynthate transport in stems of Phaseolus vulgaris L. treated with gibberellic acid,indole-3-acetic acid or kinetin.

Gibberellic acid (GA₃), indole-3-acetic acid (IAA) or kinetin (⁶N-furfurylaminopurine) applied to the apical regions of decapitated stems of derooted Phaseolus vulgaris plants, promoted ¹⁴C-photosynthate transport to the site of hormone application. Hormonal promotion of acropetal photosynthate transport was associated with significant increases in the pool size of free-space sugars at the hormone-treated region of the stem. The hormone-induced increases in the free-space pool size depended on continued phloem transport in the stem stumps while photosynthate leakage from the sink tissues of the stems was unaffected by the hormone treatments. On the basis of these observations, it is concluded that the increases in the pool size of sugars in the stem free-space results from hormonal action on processes that determine rates of sugar unloading from the sieve element-companion cell (se-cc) complexes. Furthermore, since loading of the se-cc complexes in the stem stumps was stimulated by GA₃ and IAA and unaffected by kinetin applied at the loading site, hormonal effects on net unloading from the se-cc complexes must be caused by alterations in the efflux component. For winter-grown plants, it was found that predicted increases in sugar transfer through the stem free-space from the se-cc complexes to the sink tissues could account for the observed hormonal stimulation of photosynthate transport. In contrast, for summer-grown plants the higher sugar concentrations in the stem free-space of control plants approached saturation for the sugar-accumulation process. This caused an attenuation of the responsiveness of sugar accumulation by the stem sink tissues to hormone-induced increases in the pool size of sugars in the stem free-space. On this basis it is proposed that the bulk of photosynthates may move radially from the se-cc complexes through the stem symplast of summer-grown plants.     

Photosynthesis of Ulva sp: III. O(2) Effects, Carboxylase Activities, and the CO(2) Incorporation Pattern

Ulva, a common green seaweed, performs at the biochemical level as a typical C₃ plant. Over 90% of label was found in glycerate 3-phosphate following a 3 second ¹⁴C pulse in the light, and the label was subsequently transferred to sugars. Also, the level of ribulose-1,5-bisphosphate carboxylase activity in crude extracts was about 10 times higher than that of phosphoenolpyruvate carboxylase. Concerning gas exchange, photosynthetic rates of Ulva showed no O₂ sensitivity, indicating that photorespiratory CO₂ losses are repressed as in C₄ plants. This apparent anomaly could be explained by the efficient HCO₃⁻ uptake system of Ulva which might concentrate CO₂ to the chloroplasts, thus suppressing the oxygenase activity of ribulose-1,5-bisphosphate carboxylase.