Glucose transport in thymocyte plasma-membrane vesicles

Calf-thymocyte membrane vesicles, prepared by hypotonic lysis and homogenization, were isolated by standard centrifugal techniques designed for enrichment of plasma membrane. At 20°C, these vesicles equilibrated with d-glucose and 3-O-methyl-d-glucose more rapidly than with l-glucose. About 25% of the equilibrium d-sugar space (6 μl/mg protein) was very slowly penetrated by l-glucose ( ). The time course of d-sugar accumulation in excess of l-glucose accumulation indicated that this space equilibrated with d-glucose and 3-O-methyl-d-glucose with half-times of approximately 0.2–0.4 min. The remainder of the equilibrium d-sugar space (about 75%) appeared equally accessible to both glucose isomers ( to 5 min). This was confirmed in studies of efflux from preloaded vesicles, where the d-glucose space fell with a short half-time (0.2 min) to the l-glucose space, after which the two isomers exited with the same half-time. Addition of sucrose to increase osmolarity reduced both spaces (specific and non-specific) in a manner which indicated that little if any of the vesicle sugar was bound. This was confirmed by the fact that equilibrium glucose space was independent of glucose concentration and by the fact that vesicles immediately lost their sugar when diluted with water at 0°C. These data indicate the presence of two vesicle types, discriminant and indiscriminant as regards transport of the glucose isomers. Entry of d-glucose into the discriminant (stereospecific) vesicles was temperature sensitive (Q₁₀ > 2), saturable (K_m 2 mM), and was inhibited by phloretin (K_i < 200 μM), N-ethylmaleimide (K_i < 10 mM) and cytochalasin B (K_i < 2 μM), suggesting that these vesicles contain the plasma-membrane glucose carrier. Entry of l- and d-glucose into the indiscriminant vesicles showed none of these properties. The equilibrium-exchange K_m and V were about five times the entry K_m and V, indicating the substrate loading greatly facilitates carrier translocation, at least in the outward direction.     

Evidence for negative cooperativity in human erythrocyte sugar transport

1. When d-glucose exchange influx is measure over a wide range of concentrations then two affinity constants (2.27 and 26.0 mM) are evident. This is consistent with a transport model (the allosteric pore model) in which there is negative cooperativity between subunits of the transport protein. 2. The equations for the allosteric pore model interacting with two substrates (or a substrate and an inhibitor) have been derived and have been used to analyse data from exchange inhibition and for mixed infinite-trans uptake experiments. 3. The exchange inhibition of tracer 3-O-methyl-d-glucose, d-xylose and d-fructose uptake by d-glucose also shows evidence for negative cooperativity and for two inhibition constants which are approximately equal to the d-glucose equilibrium exchange affinity constants. 4. The uptake of d-glucose into infinite-transd-glucose or 3-O-methyl-d-glucose gives K_m values of 2.6 and 2.33 mM, respectively. The uptake of 3-O-methyl-d-glucose into infinite-transd-glucose or 3-O-methyl-d-glucose gives K_m values of 6.0 and 4.6 mM, respectively. V values are slightly higher when the internal sugar is 3-O-methyl-d-glucose. 5. In cells that are treated with fluorodinitrobenzene the apparent K_i value for d-glucose inhibition of tracer d-fructose uptake is lowered. It is proposed that this is due to a partially selective effect of FDNB on the internal subunit interface stability constant (the internal pore gate).     

In Vitro Sugar Transport in Zea mays L. Kernels : II. Characteristics of Sugar Absorption and Metabolism by Isolated Developing Embryos

In vitro sugar transport into developing isolated maize embryos was studied. Embryo fresh and dry weight increased concomitantly with endogenous sucrose concentration and glucose uptake throughout development. However, endogenous glucose and fructose concentration and sucrose uptake remained constant. The uptake kinetics of radiolabeled sucrose, glucose, and fructose showed a biphasic dependence on exogenous substrate concentration. Hexose uptake was four to six times greater than sucrose uptake throughout development. Carbonylcyanide-m-chlorophenylhydrazone and dinitrophenol inhibited sucrose and glucose uptake significantly, but 3-O-methyl glucose uptake was less affected. The uptake of 1 millimolar sucrose was strongly pH dependent while glucose was not. Glucose and fructose were readily converted to sucrose and insoluble products soon after absorption into the embryo. Thus, sucrose accumulated, while glucose pools remained low. Based on the findings of this and other studies a model for sugar transport in the developing maize kernel is presented.     

Sugar Transport and Sulfite Action in Etioprotoplasts,Semi-Etioprotoplasts and Green Protoplasts of Avena sativa L.

The mechanism of glucose and sucrose transport and the influence of various concentrations of sulfite on its activity was studied in mesophyll protoplasts (etioprotoplasts, semi-etioprotoplasts and green protoplasts) isolated from oat (Avena sativa L.) seedlings. Kinetic analysis of [¹⁴C] glucose loading (in darkness) revealed in each kind of protoplasts the presence of two transport components. At low exogenous glucose concentrations a saturable system was the main mode of transport. At concentrations higher than 20 mM the loading of glucose in all types of protoplasts was dominated by a non-saturable, linear diffusion-like component. The rate of glucose uptake was greatest in etioprotoplasts and lowest in green protoplasts. In contrast to the above we have not found saturable components of sucrose transport in any kind of protoplasts. The rate of its uptake was greatest in semi-etioprotoplasts. Sulfite, at a concentration of < 1.0 mM stimulated and at ≥ 1.0 mM inhibited the uptake of glucose to etioprotoplasts and semi-etioprotoplasts and inhibited that to green protoplasts at any concentration. The transport of sucrose underwent a significant inhibition in the various types of protoplasts only under the influence of 10.0 mM of sulfite ions. Inhibition of glucose uptake by sulfite was of the non-competitive type. Sulfite also affected the level of adenylic nucleotides and lowered the energy charge and ATP/ADP ratio. Intensity of sulfite uptake was significantly higher in green protoplasts than in etioprotoplasts.     

Glucose transport in membrane vesicles from Azotobacter vinelandii,: Properties of the glucose carrierin situ

The active transport of d-glucose by membrane vesicles prepared from Azotobactervinelandii strain O is coupled to the oxidation of l-malate. The glucose carrier, but not the energy coupling system of the vesicles, is induced by growth of the cells on d-glucose medium. Vesicles isolated from A. vinelandii grown in the presence of sucrose or acetate accumulate glucose at less than 7% of the rate observed for vesicles from glucose-grown cells. Nevertheless, vesicles from sucrose- or acetate-grown cells transport sucrose or calcium, respectively, in the presence of malate.The transport system expressed in vesicles from glucose-cultured cells is highly specific for d-glucose. Studies of glucose analog uptake and of the competitive effect of analogs reveal that: (i) The glucose carrier is stereospecific. (ii) The affinity of hexoses for the transport system is inversely related to the bulk of substituents on the pyranose ring, especially at the C-1 and C-2 positions, (iii) The most effective competitors, 6-deoxyglucose and 2-deoxyglucose, exhibit affinities only 10–20% that of d-glucose for the transport system, (iv) Phloretin, but not phlorizin, is a competitive inhibitor of glucose transport, having an apparent K_i of 9 μm at pH 7.0. These latter findings suggest a similarity of the glucose transport system of fxA. vinelandii and those of eukaryotes with regard to the glucose carrier.     

Sugar transport in immature internodal tissue of sugarcane: I. Mechanism and kinetics of accumulation

Transmembrane sugar transport into immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L.) is a metabolically regulated process as evidenced by its sensitivity to pH, temperature, anaerobiosis, and metabolic inhibitors. All sugars studied—glucose, fructose, galactose, sorbose, glucose 6-phosphate, 3-O-methylglucose, and 2-deoxy-d-glucose—were apparently transported via the same carrier sites since they competed with each other for uptake. External concentrations of these sugars at one-half V_max were in the range of 3.9 to 8.4 nm. Preliminary data indicated that phosphorylation may be closely associated with glucose transport. The dominant intracellular sugar after 4-hours incubation was sucrose when glucose, glucose-6-P, or fructose was the exogenously supplied sugar; but when galactose was supplied, only 28% of intracellular radioactivity was in sucrose. Sorbose, 3-O-methylglucose, and 2-deoxy-d-glucose were not metabolized. Thus, by using these analogs, transport could be studied independently of subsequent metabolism, effectively eliminating a complicating factor in previous studies.     

Substrate specificity of sugar transport by rabbit SGLT1: single-molecule atomic force microscopy versus transport studies

In the apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/d-glucose cotransporter SGLT1 plays a crucial role in selective sugar absorption and reabsorption. How sugars are selected at the molecular level is, however, poorly understood. Here atomic force microscopy (AFM) was employed to investigate the substrate specificity of rbSGLT1 on the single-molecule level, while competitive-uptake assays with isotope-labeled sugars were performed in the study of the stereospecificity of the overall transport. rbSGLT1-transfected Chinese hamster ovary (CHO) cells were used for both approaches. Evidence of binding of d-glucose to the extracellular surface of rbSGLT1 could be obtained using AFM tips carrying 1-thio-d-glucose coupled at the C1 position to a PEG linker via a vinylsulfon group. Competition experiments with monosaccharides in solution revealed the following selectivity ranking of binding: 2-deoxy-d-glucose >or= 6-deoxy-d-glucose > d-glucose > d-galactose >or= alpha-methyl glucoside; 3-deoxy-d-glucose, d-xylose, and l-glucose did not measurably affect binding. These results were different from those of competitive alpha-methyl glucoside transport assays, where the ranking of inhibition was as follows: d-glucose > d-galactose > 6-deoxy-d-glucose; no uptake inhibition by d-xylose, 3-deoxy-d-glucose, 2-deoxy-d-glucose, or l-glucose was observed. Taken together, these results suggest that the substrate specificity of SGLT1 is determined by different recognition sites: one possibly located at the surface of the transporter and others located close to or within the translocation pathway.     

Cytochalasin B inhibition of brain glucose transport and the influence of blood components on inhibitor concentration

The effect of cytochalasin B on cerebral glucose transport and metabolism was investigated in 19 isolated perfused dog brain preparations. Cytochalasin B is a potent, non-competitive inhibitor of glucose transport at the blood-brain interface. Both glucose transport into (K_i = 6.6 ± 1.9 μM) and out of the capillary endothelial cell are inhibited. The inhibition is readily reversible by perfusion with blood containing no cytochalasin B. After 2 min of exposure to 30 μM cytochalasin B, the cerebral oxygen consumption decreased by 31% probably due to decreased availability of glucose for oxidative metabolism. About one-half of the cytochalasin B that is dissolved in blood is bound to erythrocytes and other blood components while the remainder is free.     

Sugar transport in isolated corn root protoplasts

Isolated corn (Zea mays L.) root protoplasts were used to study sucrose and hexose uptake. It is found that glucose was preferentially taken up by the protoplasts over sucrose and other hexoses. Glucose uptake showed a biphasic dependence on external glucose concentration with saturable (K_m of 7 millimolar) and linear components. In contrast, sucrose uptake only showed a linear kinetic curve. Sucrose and glucose uptake were linear over a minimum of 1 hour at pH 6.0 and 1 millimolar exogenous sugar concentration. Glucose uptake showed a sharp 42°C temperature optimum, while sucrose uptake showed a lower temperature sensitivity which did not reach a maximum below 50°C. Uptake of both sugars was sensitive to several metabolic inhibitors and external pH. Differences between sucrose and glucose uptake in two different sink tissue (i.e. protoplasts from corn roots and soybean cotyledons) are discussed.     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

Specific d-glucose transport in sarcolemma vesicles

The sarcolemmal fraction prepared from rat skeletal muscle consists of osmotically active vesicles that accumulate d-glucose in preference to l-glucose, apparently by facilitated diffusion into intravesicular space. Stereospecific d-glucose uptake by these vesicles is a saturable process, inhibited by phloridzin, by cytochalasin B, and by certain sugars, and enhanced by counterflow. An additional leak pathway permits entry of both d- and l-glucose into the vesicles.Stereospecific d-glucose transport by sarcolemmal vesicles is enhanced to a small extent by insulin, provided the hormone is administered prior to cell disruption. In membranes prepared from insulin-pretreated muscle, Ca²⁺ produces a small further enhancement. Local anesthetics preferentially inhibit stereospecific d-glucose transport. Apparent uptake of both d- and l-glucose is greater when vesicles are suspended in salt solutions rather than sucrose, an effect attributed to increased functional vesicular volume.     

Sucrose and Glucose Uptake into Beta vulgaris Leaf Tissues : A Case for General (Apoplastic) Retrieval Systems

Concentration curves for sugar and amino acid uptake by Beta vulgaris L. leaf tissues contained both a saturable and a linear component. Similarly shaped curves were obtained for influx of sucrose, glucose, and 3-O-methyl glucose by leaf discs, whole petiole slices, petiole segments containing pith tissue only, and petiole segments containing vascular bundles, although the tissues took up the various sugars via different proportions of saturable versus linear uptake. Two millimolar p-chloromercuribenzenesulfonic acid selectively inhibited the saturable component of sucrose uptake, but had almost no effect on the linear component. Uptake of glucose and 3-O-methyl glucose remained unaffected by p-chloromercuribenzenesulfonic acid treatment. Anoxia was found to inhibit the linear component of both sucrose and 3-O-methyl glucose influx, while the saturable component remained unaffected. The linear component of sucrose uptake was also competitively inhibited by maltose, as well as being selectively promoted by certain exposures to 5 millimolar N-ethylmaleimide, 2 micrograms per milliliter cycloheximide, and high levels of mannitol acting as osmoticum. These results support the proposal that the linear component is due to a process more complex than simple, or exchange, diffusion. It would also appear that the linear transport component utilizes a separate energy source than does the saturable component of sucrose influx.

Evidence for phloem loading from the apoplast was re-examined with respect to the present findings. Saturable sucrose uptake by minor vein tissues may represent retrieval of solute from the free space, which could explain the `apoplastic loading' phenomenon.

     

Characterization of the active sucrose transport system of immature soybean embryos

Immature soybean embryos were isolated from soybean [Glycine max (L.) Merr.] seeds at various stages of development to study their accumulation of [¹⁴C]sucrose in vitro. Isolated embryos accumulate sucrose at a constant rate over several hours, the label entering large, endogenous pools of sucrose from which starch, protein, and lipid storage products are formed. Accumulation is without extracellular sucrose hydrolysis and occurs predominantly by active transport at physiological sucrose concentrations. A nonsaturable diffusion component, apparently superimposed upon the active saturable component, dominates overall uptake at exogenous concentrations greater than approximately 50 millimolar sucrose. Active transport is sensitive to uncoupling agents and the sulfhydryl-modifying reagent p-chloromecuribenzene sulfonate, is dependent on more than one energy source, and exhibits well-defined requirements for incubation temperature, pH, and oxygen availability. Under optimal incubation conditions of 35°C, saturating illumination (pH 6), and 21% oxygen, the apparent K_m for sucrose is approximately 8 millimolar and V_max is approximately 0.6 micromoles per hour per 100 milligrams fresh weight. Embryos readily accumulate sucrose from dilute exogenous solutions and, when preloaded with large amounts of sucrose, maintain the internal sucrose pool against steep outward gradients. These and other observations indicate that, although perhaps fully saturated in vivo, active sucrose transport is a significant component of photosynthate uptake in developing soybean embryos, enhancing uptake at physiological sucrose concentrations 2- to 5-fold over diffusion alone.     

Taenia crassiceps: absorption of hexoses and partial characterization of Na + -dependent glucose absorption by larvae

The uptake of ¹⁴C-fructose by T. crassiceps larvae was linear with respect to concentration. Uptake of 0.05 mM¹⁴C-fructose was not inhibited by 5.0 mM unlabeled fructose, tagatose, or sorbose. Fructose appears to enter larvae by diffusion only. The uptake of radioglucose and radiogalactose was not linear with respect to concentration at low substrate concentrations; at high substrate concentrations, the uptake of both hexoses was linear with respect to concentration. Inhibitor studies indicated that both glucose and galactose enter larvae by a combination of diffusion and a mediated process, and that these hexoses are mutually competitive inhibitors of one another. The uptake of glucose and galactose was also inhibited by α-and β-methyl glucoside, fucose, and phlorizin, but not by several amino acids, certain sugar analogs, nor ouabain. Glucose transport is Na⁺ sensitive; K⁺ was demonstrated to be a competitive inhibitor of Na⁺ activation of glucose uptake. After a 90-min incubation in 5 mM unlabeled glucose, larvae accumulated glucose against an apparent concentration difference. Although larvae appear freely permeable to ouabain, this compound had no apparent effect on glucose accumulation. The results of this study are compared with previous studies on Hymenolepis diminuta, Calliobothrium verticillatum, Hydatigera (Taenia) taeniaeformis, and mammalian systems.     

Sucrose transport and hydrolysis inRhizobium tropici

TheRhizobium tropici strain CFN 299 was maintained on PY medium and was grown in minimal medium (MM) with sucrose, glucose, fructose and glutamate (or their combination) as carbon sources. Bacteria were able to simultaneously use different carbon sources and, with a combination sucrose and glutamate, the growth rate was faster than with either carbon source alone. Sucrose transport was induced by sucrose and partially repressed by glucose and glutamate if they were included in MM as additional carbon sources. The transport of sucrose was active because both an uncoupler (dinitrophenol, DNP) and inhibitors of terminal oxidation (KCN, NaN₃) severely reduced sucrose uptake. Sucrose transport was also sensitive to a functional sulfhydryl reagent but was much less sensitive to EDTA and arsenate. We obtained nonlinear Lineweaver-Burk plots for the uptake of sucrose (by sucrose-grown bacteria), and this implied the existence of at least two uptake mechanisms. Invertase (EC 3.2.1.26) is the main enzyme for sucrose hydrolysis in this organism. This enzyme was induced by sucrose and had high activity in mid-log phase cells when sucrose was the sole carbon source (0.2%). Invertase activity was not detected in growth medium. In general, the results obtained support the idea, thatR. tropici is adapted to sucrose utilization and to multicarbon nutrition during its interaction with plants.     

Host-Pathogen Interactions : XXIII. The Mechanism of the Antibacterial Action of Glycinol, a Pterocarpan Phytoalexin Synthesized by Soybeans

The biochemical basis for the ability of the pterocarpan phytoalexin glycinol (3,6a,9-trihydroxypterocarpan) to inhibit the growth of bacteria was examined. Glycinol at bacteriostatic concentrations (e.g. 50 micrograms per milliliter) inhibits the ability of Erwinia carotovora to incorporate [³H]leucine, [³H]thymidine, or [³H]uridine into biopolymers. Exposure of Escherichia coli membrane vesicles to glycinol at 20 micrograms per milliliter results in inhibition of respiration-linked transport of [¹⁴C]lactose and [¹⁴C]glycine into the vesicles when either d-lactate or succinate is supplied as the energy source. The ability of E. coli membrane vesicles to transport [¹⁴C]α-methyl glucoside, a vectorial phosphorylation-mediated process, is also inhibited by glycinol at 20 micrograms per milliliter. Furthermore, exposure of membrane vesicles to glycinol (50 micrograms per milliliter) at 20°C results in the leakage of accumulated [¹⁴C]α-methyl glucoside-6-phosphate. The effects of the phytoalexins glyceollin, capsidiol, and coumestrol, and daidzein, a compound structurally related to glycinol but without antibiotic activity, upon the E. coli membrane vesicle respiration-linked transport of [¹⁴C]glycine and of [¹⁴C]α-methyl glucoside was also examined. Glyceollin and coumestrol (50 micrograms per milliliter), but not daidzein, inhibit both membrane-associated transport processes. These data imply that the antimicrobial activity of glycinol, glyceollin, and coumestrol are due to a general interaction with the bacterial membrane. Capsidiol (50 micrograms per milliliter) inhibits d-lactate-dependent transport of [¹⁴C]glycine but not vectorial phosphorylation-mediated transport of [¹⁴C]α-methyl glucoside. Thus, capsidiol's mechanism of antimicrobial action seems to differ from that of the other phytoalexins examined.     

High-affinity transport and phosphorylation of 2-deoxy-D-glucose in synaptosomes

2-Deoxy-d -glucose (2 DG) entered synaptosomes (from rat brain) by a high-affinity, Na⁺-independent glucose transport system with a K_m, of 0.24 mM. 3-O-methyl-glucose, D-glucose, and phloretin were competitive inhibitors of 2-DG transport with K_i's of 7 mM, 64 μM, and 0·75 μM, respectively. Insulin was without effect. 2-DG uptake was also saturable at high substrate concentrations with an apparent low affinity K_m, of 75 mM, where the K_l, for glucose was 17.5 mM. We are not certain whether the rate-limiting step for the low-affinity uptake system is attributable to transport or phosphorylation. However, the high-affinity glucose transport system probably is a special property of neuronal cell membranes and could be useful in helping to distinguish separated neurons from glial cells.     

Transport of sugars by the fat body of the silkworm, Bombyx mori

The transport of glucose and α-methyl glucoside into the fat body of the silkworm, Bombyx mori L., has been studied. Glucose is transported into the tissue by a mechanism similar to facilitated diffusion and α-methyl glucoside by a diffusion process. The uptake of these sugars is neither energy dependent nor coupled to a phosphotransferase system.     

UPTAKE OF ORGANIC SUBSTRATES BY CYCLOTELLA CRYPTICA (BACILLARIOPHYCEAE): EFFECTS OF IONS,IONOPHORES AND METABOLIC AND TRANSPORT INHIBITORS1,2

The uptake of glucose and amino acids by the euryhaline diatom Cyclotella cryptica Reimann, Lewin & Guillard does not appear to be related to proton gradients. Instead, the transport systems for these organic solutes show a strong requirement for the presence of NaCl. The relationship between uptake and NaCl concentration is hyperbolic, with optimal uptake rates being approached at 100 mM NaCl. High concentrations of KCl cause strong reductions in uptake rates. The (Na⁺, K⁺)-stimulated ATPase inhibitor ouabain has no effect on glucose uptake, whereas the diphenolic glucoside phlorizin and its aglucone phloretin are strongly inhibitory. The proton translocating uncoupler CCCP (carbonylcyanide m-chlorophenyl hydrazone) and the ATPase inhibitor DCCD (dicyclohexylcarbodiimide) both almost completely abolish glucose transport, and low concentrations of the ionophares monensin and valenomycin strongly inhibit glucose uptake by the diatom. The requirement of high external NaCl concentrations for glucose transport, and the inhibitory effect an transport of the Na⁺-specific ionophore monensin are consistent with a coupling of Na⁺ and organic substrate transport, but could also be explained by a Na⁺ requirement for glucose binding to a transport carrier, and/or a possible interference with energy producing reactions associated with a monensin-induced collapse of the normal Na⁺ gradient.