Uptake of guanine by synchronizedChlorella fusca

1. The transport of guanine in autospores of light-dark synchronizedChlorella fusca was studied using radioactive guanine in the concentration range of 4 nM to 50 μM.
2. The transport system was constitutive, it had high specificity for the permeant, and theQ ₁₀ value was in the range of 1.5 to 2.2. At concentrations lower than 0.2 μM the half saturating constant, S_0.5 was 1 μM both for cells kept in dark and cells kept in light. At higher concentrations the S_0.5 of darkened cells was about 0.23 μM, while that of illuminated cells was unchanged. Only above 0.2 μM guanine did illumination of the cells or addition of glucose increase the transport rate.
3. Guanine which had accumulated did not leak out at temperatures below 45°C or by treatment with 10 μM dinitrophenol, which completely inhibited transport. Furthermore, the accumulated guanine did not exchange with exogenous guanine.
4. The guanine accumulated, more than 10⁵-fold over the external concentration, showing that the transport, was active.
5. The initial transport rate per cell revealed annual fluctuations.

     

Non-competitive inhibition of myo-inositol transport in cultured bovine retinal capillary pericytes by glucose and reversal by Sorbinil

myo-Inositol transport by retinal capillary pericytes in culture was characterized. The major myo-inositol transport process was sodium-dependent, ouabain-sensitive, and saturable at 40 mM, indicating a carrier-mediated process. The sodium ion concentration required to produce one-half the maximal rate of myo-inositol uptake ([Na+]0.5) did not show dependence on the external myo-inositol concentration (22.3 mM sodium for 0.005 mM myo-inositol; 18.2 mM sodium for 0.05 mM myo-inositol). myo-Inositol transport was an energy-dependent, active process functioning against a myo-inositol concentration gradient. The kinetics of the sodium-dependent system fitted a 'velocity type' co-transport model where binding of sodium ion to the carrier increased the velocity (Vmax 28 to 313 pmol myo-inositol/micrograms DNA per 20 min when [Na+] varied from 9 to 150 mM) but not the affinity for myo-inositol (Km 0.92 to 0.83 mM when [Na+] varied from 9 to 150 mM). Metabolizable hexoses (D-glucose or D-galactose; greater than 5 mM) inhibited myo-inositol uptake. Dixon-plot analysis indicated that the inhibition was non-competitive with a Ki of 22.7 mM for D-glucose and 72.6 mM for D-galactose. The inhibition was significantly reversed by Sorbinil (0.1 mM), an aldose reductase inhibitor. In contrast, high concentrations of non-metabolizable hexoses (L-glucose, 3-O-methyl-D-glucose), or partially metabolizable 2-deoxy-D-glucose, did not significantly inhibit myo-inositol uptake. The inhibitory effect of D-glucose or D-galactose on myo-inositol transport appeared to be related to glucose or galactose metabolism via the polyol pathway.     

Kinetic properties of the fructose influx across the brush border of the rat jejunum. Effects of a diet rich in fructose]

The unidirectional influx (i.e. initial rate of uptake) of D-fructose across the brush border of rat jejunum is a saturable function of concentration, with a Kt of 125 mM, which implicates a carrier mechanism. This mechanism appears to be very specific for fructose in view of the lack of influx inhibition observed in the presence of large concentrations of the sugars or polyols, D-glucose, D-galactose, D-mannose, D-xylose, L-sorbose, D-tagatose, sorbitol or mannitol. D-Fructose uptake is inhibited by incubation, preceded by a 30-min preincubation in the same inhibitory conditions, in the absence of Na, or in the presence of metabolic poisons, NaF, 2,4-dinitrophenol, monoiodoacetate. Phloridzin (10-3 M), with or without preincubation, has no effect on uptake. D-Fructose influx is stimulated by fructose feeding, mainly because the augmentation of the number of active sites of transfer: Jmax is increased two-fold, Kt is more weakly affected.     

Expression of Na(+) / D-glucose cotransport in Xenopus laevis oocytes by injection of poly(A)(+) RNA isolated from lobster (Homarus americanus) hepatopancreas

Xenopus laevis oocytes were used for expression and characterization of lobster (Homarus americanus) hepatopancreas Na(+)-dependent D-glucose transport activity. Poly(A)(+) RNA from the whole hepatopancreatic tissue was injected and transport activity was assayed by alpha-D-[2-(3)H] glucose. Injection of lobster hepatopancreatic poly(A)(+) RNA resulted in a dose (1-20 ng) and time (1-5 days) dependent increase of Na(+)-dependent D-glucose uptake. Kinetics of Na(+)-dependent glucose transport was a hyperbolic function (K(m)=0.47+/-0.04 mM) of external D-glucose concentration and a sigmoidal function (K(Na)=68.32+/-1.57 mM; Hill coefficient=2.22+/-0.09) of external Na(+) concentration. In addition, Na(+)-dependent D-glucose uptake was significantly inhibited by both (0.1-0.5 mM) phloridzin and (0.1-0.5 mM) methyl-alpha-D-glucopyranoside. After size fractionation through a sucrose density gradient, poly(A)(+) RNA fractions with an average length of 2-4 kb induced a twofold increase in Na(+)-dependent phloridzin-inhibited D-glucose uptake as compared to total poly(A)(+) RNA-induced uptake. The results of this study provide the functional basis to screen lobster hepatopancreatic cDNA libraries for clones encoding putative and still not known crustacean SGLT-type Na(+)/glucose co-transporter(s).     

Kinetics of iron absorption by excised rice roots

Summary Studies on the rate of iron absorption by excised rice roots from solutions of different concentrations of FeSO₄ showed the presence of two patterns, one in the low (0.005–0.5 mM) and the other in the high (1–30 mM) concentration range. The presence of CaSO₄ or MnSO₄ at 0.5 mM enhanced Fe⁺⁺ absorption in the low concentration range, while CaSO₄ at 10 mM inhibited Fe absorption in the high concentration range in a competitive manner. Fe⁺⁺ absorption at both low and high concentrations was sensitive to metabolic inhibitors. The isotherm for Fe⁺⁺ absorption at O° exhibited an initial absorption shoulder in both low and high concentrations and was suggestive of a latent ion-transport capacity for Fe⁺⁺ in rice roots.     

Kinetics and stoichiometry of Na-dependent Li transport in human red blood cells

This paper describes the kinetics and stoichiometry of a tightly coupled Na-Li exchange transport system in human red cells. The system is inhibited by phloretin and furosemide but not by ouabain. Li influx by this system increases and saturates with increasing concentrations of external Li and internal Na and is inhibited competitively by external Na. Comparable functions relate Li efflux and Na efflux to internal and external Li and Na concentrations. Analysis of these relations yields the following values for the ion concentrations required to half-maximally activate the transport system: internal Na and Li 9.0 and 0.5 mM, respectively, external Na and Li 25 and 1.5 mM, respectively. The system performs a 1:1 exchange of Na and Li moving in opposite directions across the red cell membrane. We found no evidence for a simultaneous transport of more than one Na and Li by the system. The maximum transport rate of Na-dependent Li transport varied between 0.1 and 0.37 mmol/(liter of cells X h) in the red cells of the five normal male subjects studied. No significant variations between individual subjects were observed for bicarbonate-stimulated Li transport and for the residual Li fluxes which occur in the absence of bicarbonate and in the presence of ouabain plus phloretin.     

Interaction of facilitative glucose transporter with glucokinase and its modulation by ADP and glucose-6-phosphate

Bacterial glucokinase (GK) binds to purified, human erythrocyte glucose transporter (GT) reconstituted in vesicles. The binding is largely abolished if GT is predigested with trypsin, indicating that GK binds to the cytoplasmic domain of GT. The binding is a saturable function of GK concentration showing two distinct affinities with apparent K_D of 0.33 and 5.1 μM. The binding is stimulated by an increasing concentration of ADP with the 50% maximal effect at 5 mM. Glucose-6-phosphate (G6P) also stimulates the binding with a distinct optimum at 25 mM. The binding is stimulated only slightly by ATP. D-glucose has no affect on the binding. KCl enhances the binding with the maximal effect at physiological intracellular concentrations. The binding is sensitive to changes in pH with an optimum at pH 4. The binding causes no detectable functional change in GT. However, the enzymatic activity of GK measured at nanomolar concentrations of GK is significantly greater in the presence of GT vesicles than in its absence or in the presence of protein-free vesicles, indicating that GK interacts with GT at this low concentration range with an apparent K_D of 10 mM. Although its physiological significance is not known, the GK-GT interaction in vitro described here suggests that these two proteins may also interact in the cell and regulate carbohydrate metabolism. © 1993 Wiley-Liss, Inc.     

Interactions of sodium pentobarbital with D-glucose and L-sorbose transport in human red cells.

Pentobarbital acts as a mixed inhibitor of net D-glucose exit, as monitored photometrically from human red cells. At 30 degrees C the Ki of pentobarbital for inhibition of Vmax of zero-trans net glucose exit is 2.16+/-0.14 mM; the affinity of the external site of the transporter for D-glucose is also reduced to 50% of control by 1. 66+/-0.06 mM pentobarbital. Pentobarbital reduces the temperature coefficient of D-glucose binding to the external site. Pentobarbital (4 mM) reduces the enthalpy of D-glucose interaction from 49.3+/-9.6 to 16.24+/-5.50 kJ/mol (P<0.05). Pentobarbital (8 mM) increases the activation energy of glucose exit from control 54.7+/-2.5 kJ/mol to 114+/-13 kJ/mol (P<0.01). Pentobarbital reduces the rate of L-sorbose exit from human red cells, in the temperature range 45 degrees C-30 degrees C (P<0.001). On cooling from 45 degrees C to 30 degrees C, in the presence of pentobarbital (4 mM), the Ki (sorbose, glucose) decreases from 30.6+/-7.8 mM to 14+/-1.9 mM; whereas in control cells, Ki (sorbose, glucose) increases from 6.8+/-1.3 mM at 45 degrees C to 23.4+/-4.5 mM at 30 degrees C (P<0.002). Thus, the glucose inhibition of sorbose exit is changed from an endothermic process (enthalpy change=+60.6+/-14.7 kJ/mol) to an exothermic process (enthalpy change=-43+/-6.2 7 kJ/mol) by pentobarbital (4 mM) (P<0.005). These findings indicate that pentobarbital acts by preventing glucose-induced conformational changes in glucose transporters by binding to 'non-catalytic' sites in the transporter.     

Transport pathways in canine lingual epithelium involved in sweet taste

The responses of canine lingual epithelium to D-glucose weremeasured in an Ussing chamber to determine the possible contributionof the osmotic changes of taste cells to the response of saccharides.With the mucosal solution containing 50 mM NaCl, 2 mM HEPES,pH 7.4 (solution A) and the serosal solution containing Krebs—Henseleit(KH) buffer the addition of up to 0.5 M D-glucose in the mucosalsolution increased the short circuit current (I_sc) in a sigmoidalmanner. The D-glucose-stimulated I_sc was inhibited by 0.1 mMamiloride or 1 mM ouabain added to either the mucosal or theserosal solution, and partially inhibited by 5 mM BaCl₂ addedto the serosal solution. The inhibition by these three compoundswas also observed in the presence of 0.5 M NaCl. Ouabain alsoinhibited transport when added to solution A. These experimentssuggest that in canine lingual epithelium the paracellular pathwaypermits molecules as large as ouabain (mol. wt 586) to diffusefrom the mucosal to the serosal solution and vice versa underall osmotic conditions. These results may explain the phenomenonof intravascular taste. Such is not the case in rat tongue whereouabain only inhibited transport when added to the serosal solution.Increasing the osmolality of the serosal KH buffer by additionof relatively membrane-impermeable saccharides such as sucroseor L-glucose did not significantly alter the I_sc, whereas makingthe serosal KH solution hypo-osmotic resulted in a transientdecrease in I_sc. These data suggest that the increase in I_scinduced by saccharides, such as D-glucose, is not simply anosmotic response of the epithelium but more likely the consequenceof saccharides binding weakly to receptors. That the responseto both salts by themselves and in the presence of saccharidesexhibits the same cation selectivity, and that both are inhibitedby amiloride, ouabain, BaCl₂ and LaCl₃ suggest that in caninelingual epithelia, in contrast to rat epithelium, the responsesto hyperosmotic concentrations of salts and saccharides mightoccur via the same transcellular pathways.     

Author index

(1) Cyclic AMP stimulated alanine transport in isolated hepatocytes by approx. 30%, in the range 0.2–5 mM alanine. (2) Alanine utilisation was also stimulated by cyclic AMP. The rates of transport and metabolism were comparable, both in the presence and absence of cyclic AMP. (3) At concentrations of alanine above 1 mM, addition of ouabain, or the reduction of the Na⁺ concentration, could partially inhibit transport without affecting the rate of metabolism. (4) At these alanine concentrations, stimulation of metabolism by cyclic AMP was associated with a decrease in the intracellular to extracellular alanine concentration ratio. (5) At alanine concentrations below 0.5 mM, or at higher concentrations when transport was inhibited by reducing the Na⁺ concentration, cyclic AMP caused an increase in the alanine concentration ratio. (6) It is concluded that at concentrations of alanine above 1 mM, alanine transport is not rate-limiting for alanine metabolism in hepatocytes from fed rats, and cyclic AMP stimulates alanine metabolism primarily by an effect on an intracellular reaction. At physiological concentrations of alanine, however, alanine transport appears to be rate-limiting in agreement with a previous report.     

GLT-1 Transport Stoichiometry Is Constant at Low and High Glutamate Concentrations when Chloride Is Substituted by Gluconate

Glutamate is the major excitatory neurotransmitter, but prolonged exposure even at micromolar concentrations causes neuronal death. Extracellular glutamate is maintained at nanomolar level by glutamate transporters, which, however, may reverse transport and release glutamate. If and when the reverse occurs depends on glutamate transport stoichiometry (GTS). Previously we found that in the presence of chloride, the coupled GLT-1 glutamate transporter current and its relationship to radiolabeled glutamate flux significantly decreased when extracellular glutamate concentration increased above 0.2 mM, which implies a change in GTS. Such high concentrations are feasible near GLT-1 expressed close to synaptic release site during excitatory neurotransmission. The aim of this study was to determine GLT-1 GTS at both low (19–75 μM) and high (300–1200 μM) glutamate concentration ranges. GTS experiments were conducted in the absence of chloride to avoid contributions by the GLT-1 uncoupled chloride conductance. Mathematical analysis of the transporter thermodynamic equilibrium allowed us to derive equations revealing the number of a particular type of ion transported per elementary charge based on the measurements of the transporter reversal potential. We found that GLT-1a expressed in COS-7 cells co-transports 1.5 Na⁺, 0.5 Glu^-, 0.5 H⁺ and counter-transports 0.6 K⁺ per elementary charge in both glutamate concentration ranges, and at both 37°C and 26°C temperatures. The thermodynamic parameter Q ₁₀ = 2.4 for GLT-1 turnover rate of 19 s^-1 (37°C, -50 mV) remained constant in the 10 μM–10 mM glutamate concentration range. Importantly, the previously reported decrease in the current/flux ratio at high glutamate concentration was not seen in the absence of chloride in both COS-7 cells and cultured rat neurons. Therefore, only in the absence of chloride, GLT-1 GTS remains constant at all glutamate concentrations. Possible explanations for why apparent GTS might vary in the presence of chloride are discussed.     

Effect of glucose uptake on growth rate of mouse 3T3 cells

The objective of this investigation was to determine whether the rate of glucose uptake by mouse 3T3 cells was a primary determinant of growth rate. The experimental approach was to control the rate of glucose uptake into intracellular pools by supplying this sugar at varying concentration in minimal Eagle's medium with dialyzed serum in the absence and presence of 6-deoxy-D-glucose, a metabolically inert homomorphic analog of D-glucose that competitively inhibits the uptake of D-glucose. Total hexose (D-glucose and 6-deoxy-D-glucose) concentration was maintained at the physiological concentration of 5.5 mM, in order to maintain saturation and maximum activity of the D-glucose transport system; thus the flux of D-glucose into the cell was controlled by adjusting its concentration relative to its competing nonmetabolizable analog. It was found that even when the concentration of D-glucose was reduced to 0.7 mM, one eighth of the “normal” level of 5.5 mM. and 6-deoxy-D-glucose was present in sevenfold excess (4.8 mM), conditions under which glucose uptake was reduced to 20% of that shown by cells in the presence of 5.5 mM D-glucose, and intracellular pools of glucose and phosphorylated sugars derived from glucose were reduced to approximately 14% of normal, there was not a significant decrease in growth rate. These data support the view that the rate of glucose uptake is not a primary determinant of growth rate under the usual conditions of cell culture.     

Transport of 2-aminoisobutyric acid in mesophyll protoplasts of Pisum sativum L. Application of silicone oil layer centrifugation

Uptake of 2-amino [1-¹⁴C]isobutyric acid by pea mesophyll protoplasts was investigated using silicone oil layer centrifugation. Uptake was expressed on the basis of the ³H₂O-space of the protoplast pellet. The ³H₂O-space can be used as a measure of protoplast volume, taking into consideration that about 10% of it is extracellular space. At concentrations in the range 10 μM–10 mM, the uptake of 2-aminoisobutyric acid (Aib) was linear with time for at least 1 h. At an external concentration of 10 μM, up to a 10-fold accumulation of Aib in the protoplasts was observed during 1 h of incubation. The concentration-dependence of the uptake rate conforms to the sum of a Michaelis-Menten term and a linear term. Large differences in uptake rates were found for different preparations of protoplasts, especially at low concentrations of Aib. This could be attributed to differences in the activity of the saturable component. Both transport components were strongly inhibited by 10 μM CCCP, even when transport was apparently downhill.     

Inhibition of sugar active transport across rat intestine in vivo by cadmium

Galactose absorption by rat jejunum perfused in vivo is inhibited by 0.5 mM Cd2+. This effect is explained by impairment of the phlorizin-sensitive sugar transport system, as Cd does not modify the absorption of L-sorbose or that of galactose in the presence of 0.5 mM phlorizin. Cd inhibition is observed as early as in the 1st minute, does not increase by previous exposure of the mucosa to the metal and does not decrease after washing with saline solution, but it is entirely reversed by EDTA or dithioerythritol. Results agree with a Cd2+ binding to HS- groups of membrane proteins at the brush border, appertaining or functionally related to the phlorizin-sensitive and Na+ dependent transport system for sugars.     

Purification of rabbit liver phosphofructokinase and its properties under simulatingin vivo conditions

Phosphofructokinase (EC 2.7.1.11) from rabbit liver was purified to homogeneity. Preincubation of enzyme results in nonlinearity of enzyme activity with enzyme concentration. Therefore K_0.5 of enzyme for fructose 6 phosphate in the absence or presence of fructose 2,6 bisphosphate or polyethylene glycol or in the presence of both was determined at physiological concentrations of its various effectors by taking the initial rate obtained by adding the enzyme last. They decrease the K_0.5 value from 4.1 mM to about 0.2mM. The K_0.5 of enzyme for fructose 2,6 bisphosphate was also determined under the above conditions. It is about 4.3ΜM. Transient kinetics of phosphofructokinase at varying concentrations of enzyme in the presence of fructose 2,6 bisphosphate or polyethylene glycol or in the presence of both were studied. It was found that although they decrease t_1/2 i.e. the time to reach half the maximal steady rate by about 5–8 fold, it was about constant at varying concentrations of the enzyme. These results indicate that fructose 2,6 bisphosphate and polyethylene glycol decrease K_0.5 of the enzyme for fructose 6 phosphate not by associating the enzyme to higher aggregates, but by a different mechanism.     

Competitive substrate inhibition of amyloglucosidase from Rhizopus sp. by vanillin and curcumin

Kinetic studies of two glucosylation reactions catalyzed by an amyloglucosidase from Rhizopus sp. leading to the synthesis of vanillin-α/β-D-glucoside from D-glucose and vanillin and curcumin-bis-α-D-glucoside from D-glucose and curcumin were investigated in detail. Initial reaction rates were determined from kinetic runs involving different concentrations of D-glucose and vanillin (5?mM to 0.1?M) or D-glucose and curcumin (5?mM to 0.1?M). Graphical double reciprocal plots showed that the kinetics of the two enzyme catalyzed reactions exhibited Ping-Pong Bi-Bi mechanism where competitive substrate inhibition by vanillin/curcumin led to dead-end amyloglucosidase–vanillin/curcumin complexes at higher concentrations of vanillin/curcumin. An attempt to obtain the best fit of this kinetic model through computer simulation yielded in good approximation, the values of four important kinetic parameters, vanillin-α/β-D-glucoside: k_cat=35.0±3.2 10^?5M?h^?1·mg, K_i=10.5±1.1?mM, K_mD-glucose=60.0±6.2?mM, K_mvanillin=50.0±4.8?mM; curcumin-bis-α-D-glucoside: k_cat=6.07±0.58 10^?5M?h^?1·mg, K_i=3.0±0.28?mM, K_mD-glucose=10.0±0.9?mM, K_mcurcumin=4.6±0.5?mM.     

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.     

Characterization of a glucose transport system in Vibrio parahaemolyticus.

Cells of a glucose-PTS (phosphoenolpyruvate:carbohydrate phosphotransferase system)-negative mutant of Vibrio parahaemolyticus transport D-glucose in the presence of Na+. Maximum stimulation of D-glucose transport was observed at 40 mM NaCl, and Na+ could be replaced partially with Li+. Addition of D-glucose to the cell suspension under anaerobic conditions elicited Na+ uptake. Thus, we conclude that glucose is transported by a Na+/glucose symport mechanism. Calculated Vmax and Km values for the Na(+)-dependent D-glucose transport were 15 nmol/min/mg of protein and 0.57 mM, respectively, when NaCl was added at 40 mM. Na+ lowered the Km value without affecting the Vmax value. D-Glucose was the best substrate for this transport system, followed by galactose, alpha-D-fucose, and methyl-alpha-glucoside, judging from the inhibition pattern of the glucose transport. D-Glucose itself partly repressed the transport system when cells were grown in its presence.     

Transport of glucose and fructose in rat hepatocytes at 37 degrees C

The kinetic parameters for transport of the nonmetabolizable glucose analogue 3-O-methyl-D-glucose and the relationship between transport and metabolism of D-glucose and D-fructose were determined in isolated rat hepatocytes at 37 degrees C and pH 7.4. 3-O-Methylglucose at a very low concentration (0.1 mM) equilibrated with the intracellular water with a rate constant of 0.41 s-1. Km for equilibrium exchange entry was 5.5 mM and Vmax was 2.2 mM X s-1 and similar results were obtained when using the zero-trans entry protocol. The rate constant for entry of tracer D-glucose was 0.15 s-1 and Km for glucose was about 20 mM. The phosphorylation rate for D-glucose was much slower than the transport rate. The rate constant for D-fructose entry was about 0.04 s-1, the apparent Km was about 100 mM and Vmax about 5 mM X s-1. The concentration dependence of 3-O-methylglucose inhibition of labelled fructose transport revealed biphasic kinetics indicating that fructose was transferred by both the glucose transporter and a fructose transporter. At concentrations lower than 1 mM, fructose metabolism appeared to be limited by the transport step.     

On the mechanism of sugar and amino acid interaction in intestinal transport.

The influence of amino acids on D-glucose transport was studied in isolated vesicles of brush border membrane from rat small intestine. It is demonstrated that: (a) Uptake of D-glucose by the membranes is inhibited by simultaneous flow of L- and D-alanine into the vesicles. (b) Addition of L-alanine to membranes pre-equilibrated with D-glucose causes efflux of this sugar. (c) The influence of amino acids on D-glucose is dependent on the presence of Na+. (d) The ionophorous agents monactin and valinomycin are able to prevent the transport interaction of D-glucose and amino acids. Monactin is effective in the presence of Na+ without further addition of other cations, while valinomycin is effective only with added K+, in accordance with the known specificity of these antibiotics. (e) The inhibitory effect increases with L-alanine concentration up to about 50 mM after which it levels off. The experiments provide evident that the Na+-dependent sugar and amino acid fluxes across the brush border membrane are coupled electrically.