Transport of sugars in chick-embryo fibroblasts. Evidence for a low-affinity system and a high-affinity system for glucose transport.

The rate of D-glucose uptake by cells that had been deprived of sugar for 18-24h was consistently observed to be 15-20 times higher than that in control cells maintained for the same length of time in medium containing glucose. This increased rate of glucose transport by sugar-starved cells was due to a 3-5-fold increase in the Vmax. value of a low-affinity system (Km 1 mM) combined with an increase in the Vmax of a separate high-affinity system (Km 0.05-0.2 mM). The high-affinity system, which was most characteristic of starved cells, was particularly sensitive to low concentrations of the thiol reagent N-ethylmaleimide; 50% inhibition of uptake occurred at approx. 0.01 mM-N-ethylmaleimide. In contrast with the high-affinity system, the low-affinity system of either the fed cells or the starved cells was unaffected by N-ethylmaleimide. In addition to the increases in the rate of D-glucose transport, cells deprived of sugar had increased rates of transport of 3-O-methyl-D-glucose and 2-deoxy-D-glucose. No measurable high-affinity transport system could be demonstrated for the transport of 3-O-methylgucose, and N-ethylmaleimide did not alter the initial rate. Thus the transport of 3-O-methyglucose by both fed and starved cells was exclusively by the N-ethylmaleimide-insensitive low-affinity system. The low-affinity system also appeared to be the primary means for the transport of 2-deoxyglucose by fed and starved cells. However, some of the transport of 2-deoxyglucose by starved cells was inhibited by N-ethylmaleimide, suggesting that 2-deoxyglucose may also be transported by a high-affinity system. The results of experiments that measured transport kinetics strongly suggest that glucose can be transported by a least two separate systems, and 3-O-methylglucose and 2-deoxyglucose by one. Support for these interpretations comes from the analysis of the effects of N-ethylmaleimide and cycloheximide as well as from the results of competition experiments. The uptake of glucose is quite different from that of 2-deoxyglucose and 3-O-methylglucose. The net result of sugar starvation serves to emphasize these differences. The apparent de-repression of the transport systems studied presents an interesting basis for further studies of the regulation of transport in a variety of cells.     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H(+) -dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of D- and L-[U-(14)C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-D-glucose/D-[U-(14)C]glucose and 3-O-methyl-D-glucose/3-O-methyl-D-[U-(14)C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH(4)Cl inhibited neither the linear component of D- and L-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-D-[U-(14)C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol(-1), respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol(-1)). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Proline and valine transport in Agrobacterium tumefaciens C-58

Abstract The transport of proline and valine by Agrobacterium tumefaciens was investigated. Proline uptake by starved cells was found to be higher in the presence of glucose than in its absence. Valine uptake, on the other hand, was lowered in the presence of glucose. Proline uptake was found to be insensitive to the action of dicyclohexyl carbodiimide (DCCD), a membrane ATPase inhibitor, whereas valine uptake appeared to show some sensitivity to this agent. The results obtained seem to suggest that the two transport systems make use of different energy stores.     

Energetics underlying the process of long-chain fatty acid transport.

The gram negative bacterium Escherichia coli has evolved a highly specific system for the transport of exogenous long-chain fatty acids (C12-C18) across the cell envelope that requires the outer membrane protein FadL and the inner membrane associated fatty acyl CoA synthetase. The transport of oleate (C18:1) across the cell envelop responds to metabolic energy. In order to define the source of metabolic energy which drives this process, oleate transport was measured in wild-type and ATP synthase-defective (Deltaatp) strains which were (i) subjected to osmotic shock and (ii) starved and energized with glucose or d-lactate in the presence of different metabolic inhibitors. Osmotic shock did not eliminate transport but rather reduced the rate to 33-55% of wild-type levels. These results suggested a periplasmic protein may participate in this process or that osmotic shock disrupts the energized state of the cell which in turn reduces the rate of oleate transport. Transport systems which are osmotically sensitive also require ATP. The process of long-chain fatty acid transport requires ATP generated either by substrate-level or oxidative phosphorylation. Following starvation, the basal rate of transport for wild-type cells was 340.4 pmol/min/mg protein compared to 172.0 pmol/min/mg protein for the Deltaatp cells. When cells are energized with glucose, the rates of transport were increased and comparable (1242.6 and 1293.8 pmol/min/mg protein, respectively). This was in contrast to cells energized with d-lactate in which only the wild-type cells were responsive. The role of ATP is likely due to the ATP requirement of fatty acyl CoA synthetase for catalytic activity. The process of oleate transport is also influenced by the energized state of the inner membrane. In the presence of carbonyl cyanide-m-chlorophenylhydrazone oleate transport is depressed to 30-50% of wild-type levels in wild-type and Deltaatp strains under starvation conditions. These results are mirrored in cells energized with glucose and d-lactate, indicating that an energized membrane is required for optimal levels of oleate transport. These data support the hypothesis that the fatty acid transport system of E. coli responds to both intracellular pools of ATP and an energized membrane for maximal proficiency.     

Effect of Glucose Starvation on Glucose Transport in Neuronal Cells in Primary Culture from Rat Brain

The regulation of glucose transport into cultured brain cells during glucose starvation was studied. On glucose deprivation for 40 h, 2-deoxy-D-glucose (2-DG) uptake was stimulated twofold in neuronal cells but was not changed significantly in astrocytes. On refeeding, the increased activity of neuronal cells rapidly returned to the basal level, an observation indicating that the effect of glucose starvation was reversible. The increase was due solely to change in the Vmax, a finding suggesting that the number of glucose transporters on the plasma membrane is increased in starved cells. Cycloheximide inhibited this increase. In the presence of cycloheximide, the activity of 2-DG uptake of starved cells remained constant for 12 h and then slowly decreased, whereas that of fed cells decreased rapidly. These findings suggest that glucose starvation regulates glucose transport by changing the rate of net synthesis of the transporter in neuronal cells in culture.     

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     

Methionine transport in Yersinia pestis.

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.     

Stimulation of zero-trans rates of lactose and maltose uptake into yeasts by preincubation with hexose to increase the adenylate energy charge

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabeled sugars for 5 to 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth medium, washed, suspended in nutrient-free buffer, and stored on ice before they are assayed. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae strains harvested from lactose fermentations were three- to eightfold lower than the specific rates of lactose consumption during fermentation. No significant extracellular beta-galactosidase activity was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before the [(14)C]lactose uptake reactions were started. A short (1- to 7-min) preincubation of the yeasts with 10 to 30 mM glucose caused 1.5- to 5-fold increases in the specific rates of lactose uptake. These increases correlated with increases in EC (from 0.6 to 0.9) and ATP (from 4 to 8 micromol x g dry yeast(-1)). Stimulation by glucose affected the transport V(max) values, with smaller increases in K(m) values. Similar observations were made for maltose transport, using a brewer's yeast. These findings suggest that the electrochemical proton potential that drives transport through sugar/H(+) symports is significantly lower in the starved yeast suspensions used for zero-trans assays than in actively metabolizing cells. Zero-trans assays with such starved yeast preparations can produce results that seriously underestimate the capacity of sugar/H(+) symports. A short exposure to glucose allows a closer approach to the sugar/H(+) symport capacity of actively metabolizing cells.     

Transport enhancement and reversal: glucose and 3-O-methyl glucose.

In chick embryo fibroblast cultures the 15- to 30-fold enhancement of D-glucose uptake observed when cells are starved of glucose for 24 hours is not duplicated for derivatives of glucose that compete effectively for uptake and have generally been considered to use the same carrier. 2-deoxy-D-glucose, D-mannose, D-galactose and D-glucosamine are derepressed progressively less sharply in that order with glucosamine uptake never more than doubled by starvation. D-glucose at a concentration of 5.5 mM in the 24-hour conditioning medium is a strong "repressor" resulting in low "transport" behavior for each of the five sugars cited. D-glucosamine is equally effective at the same concentration. A 10-fold reduction in the concentration of glucosamine (0.55 mM) allows for the escape from repression of mannose, glucose, and deoxyglucose uptake while the others remain repressed. Mannose uptake escapes as well when the glucose concentration in the "conditioning" medium is similarly reduced. Under certain conditions of starvation and cell density dramatic effects of supplemental stimulation by insulin can be achieved. Insulin withdrawal interrupts the supplemental stimulation process. Cycloheximide, actinomycin D and cordycepin block both non-insulin and insulin-induced derepression. Short exposure (15-30 minutes) of 24-hour starved cells to glucose (5.5 mM) reduces glucose sharply but does not affect 3-O-methyl glucose uptake. If the exposure is to 2-deoxyglucose (5.5 mM) further derepression of glucose uptake results.     

Silicon metabolism in diatoms. III. Respiration and silicon uptake in Navicula pelliculosa

1. Evidence is presented that silicon uptake in the diatom Navicula pelliculosa is linked with aerobic respiration. 2. Cyanide, fluoride, iodoacetate, arsenite, azide, and fluoroacetate, at concentrations inhibitory to respiration, were also inhibitory to silicon uptake. 3. 2,4-Dinitrophenol (1 to 2 x 10(-5)M) stimulated respiration by 100 per cent, but almost completely inhibited silicon uptake. 4. The respiratory quotient of non-Si-deficient cells decreased from 0.93 to 0.75 after 4 days of starvation in darkness. Glucose (1 per cent) raised the respiratory quotient of such starved cells to 1.05. 5. Silicate (20 mg. Si/liter) stimulated respiration of unstarved Si-deficient cells by about 40 per cent. The effect of silicate on the respiration of Si-deficient cells which had been starved in darkness for 4 days was less marked. 6. The respiratory quotient of Si-deficient cells decreased from 0.8-0.9 to 0.3 after 4 days of starvation in darkness. The addition of silicate to starved cells raised the quotient to 0.5. This represented a 25 per cent stimulation of oxygen uptake concomitant with a 90 per cent stimulation of carbon dioxide evolution. 7. Glucose (1 per cent) caused an increase of respiratory quotient in starved cells from 0.3 to 0.7-0.8. The addition of silicate had no effect on the R.Q. during the oxidation of exogenous glucose. 8. Substrates (glucose, fructose, galactose, lactate, succinate, citrate, glycerol), which caused a stimulation of respiration in starved cells, also stimulated silicon uptake by those cells. However, the stimulation of silicon uptake (50 to 100 per cent) was not proportional to the respiratory stimulation by these substrates (30 to 300 per cent).     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H⁺-dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of d- and l-[U-¹⁴C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-d-glucose/d-[U-¹⁴C]glucose and 3-O-methyl-d-glucose/3-O-methyl-d-[U-¹⁴C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH₄Cl inhibited neither the linear component of d- and l-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-d-[U-¹⁴C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol^− 1, respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol^− 1). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) porphyrin compounds

1. There was no apparent correlation between the rate of respiration and rate of accumulation of proline in Candida albicans cells. 2. In contrast to normal cells, the respiration in the starved cells became completely cyanide insensitive. The starvation of cells in the presence of cycloheximide prevented the cells from becoming cyanide insensitive. The addition of Fe(III), however, accelerated the process. 3. Oxidizable substrates e.g. NADH, acetate and glucose, when added to cyanide-insensitive starved cells, exhibited 40--280% stimulation in respiration rate. However, this enhancement in oxidation by various substrates was not coupled to a simultaneous increase in the proline uptake or in intracellular ATP levels. 4. There was 6-fold stimulation in proline uptake when cyanide-insensitive cells were preincubated with 50 mM glucose. The preincubation of starved cells resulted in a partial restoration of cyanide sensitivity and increased intracellular ATP levels. The preincubation of starved cells with other oxidizable substrates resulted in a partial restoration of cyanide sensitivity but had no stimulatory effect on intracellular ATP levels and proline accumulation. 5. Both the enhanced uptake and ATP levels in glucose preincubated cells were found to be completely abolished by iodoacetate. 6. It is proposed that the increased proline uptake in cells preincubated with glucose was mainly due to the production of glycolytic energy.     

The role of N-glycosylation of GLUT1 for glucose transport activity.

To elucidate a functional role of N-glycosylation in glucose transporters, we introduced oligonucleotide-directed mutagenesis in GLUT1 cDNA to remove the possible site for N-linked glycosylation. The wild-type and the mutated GLUT1 cDNAs which induced a mutation of Asn at residue 45 to Asp, Tyr, or Gln were transfected and stably expressed into Chinese hamster ovary cells. The expressed wild-type and the mutated GLUT1 was demonstrated to be a broad band of a 45-60-kDa form and a sharp band of a 38-kDa form on Western blot analysis, respectively, indicating no glycosylation in the mutated GLUT1. Although the cell surface labeling of the glucose transporters demonstrated the presence of the glycosylation-defective glucose transporters on the cells surface, photoaffinity labeling of glycosylation-defective GLUT1 with [3H] cytochalasin B and a photoreactive mannose derivative, [3H]2-N-4-(1-azi-2,2,2,trifluoroethyl)benzoyl-1,3-bis(D-mannos+ ++-4-yloxy)-2- propylamine in the membranes was observed to be 40-70 and 15-30% of that of the wild-type GLUT1, respectively. The kinetic study of 2-deoxyglucose uptake revealed that the glycosylation-defective GLUT1 had a 2-2.5-fold greater Km value for 2-deoxyglucose uptake compared with the wild-type GLUT1. These observations strongly suggest that 1) N-glycosylation of GLUT1 glucose transporter is only on Asn 45 and 2) N-glycosylation plays an important role in maintaining a structure of glucose transporter with high affinity for glucose, thus, with high transport activity.     

The effect of SH group inhibitors on phosphate transport in Chlorella pyrenoidosa]

The effect of Sulphydryl reagents have been investigated. pCMB inhibits the transport of phosphate in Chlorella pyrenoidosa. This inhibition is immediate and does not change as a function of time of incubation. This inhibition affects non starved and starved cells (phosphate omitted). pCMPS and Mersalyl act in the same manner as pCMB. When these compounds are used at low concentrations, inhibition of phosphate uptake is observed only in starved cells. The substrate (phosphate) cannot provide protection against this inhibition. NEM inhibits phosphate uptake and this inhibition increases as a function of time of incubation. When the time of incubation is very short (about 15 seconds) the effects seems to be superficial and NEM reacts with SH groups involved in the transport system. When phosphate is present (for 15 seconds of incubation with NEM) the inhibition is less important than when phosphate is omitted. The substrate protects against NEM, but this protection disappears as the incubation with NEM is prolonged. NEM inhibits phosphate uptake in non starved and starved cells, however, it is observed that the inhibition is less important in starved cells than in non starved cells. The authors conclude that two kinds of SH groups might exist in the phosphate transport system, one reacting with pCMB and the other with NEM.     

Characterization of the Rhizobium (Sinorhizobium) meliloti high- and low-affinity phosphate uptake systems.

Genetic studies have suggested that Rhizobium (Sinorhizobium) meliloti contains two distinct phosphate (Pi) transport systems, encoded by the phoCDET genes and the orfA-pit genes, respectively. Here we present data which show that the ABC-type PhoCDET system has a high affinity for Pi (Km, 0.2 microM) and that Pi uptake by this system is severely inhibited by phosphonates. This high-affinity uptake system was induced under Pi-limiting conditions and was repressed in the presence of excess Pi. Uptake via the OrfA-Pit system was examined in (i) a phoC mutant which showed increased expression of the orfA-pit genes as a result of a promoter-up mutation and (ii) a phoB mutant (PhoB is required for phoCDET expression). Pi uptake in both strains exhibited saturation kinetics (Km, 1 to 2 microM) and was not inhibited by phosphonates. This uptake system was active in wild-type cells grown with excess Pi and appeared to be repressed when the cells were starved for Pi. Thus, our biochemical data show that the OrfA-Pit and PhoCDET uptake systems are differentially expressed depending on the state of the cell with respect to phosphate availability.     

d-Glucose Transport System of Zymomonas mobilis

The properties of the d-glucose transport system of Zymomonas mobilis were determined by measuring the uptake of nonmetabolizable analogs (2-deoxy-d-glucose and d-xylose) by wild-type cells and the uptake of d-glucose itself by a mutant lacking glucokinase. d-Glucose was transported by a constitutive, stereospecific, carrier-mediated facilitated diffusion system, whereby its intracellular concentration quickly reached a plateau close to but not above the external concentration. d-Xylose was transported by the d-glucose system, as evidenced by inhibition of its uptake by d-glucose. d-Fructose was not an efficient competitive inhibitor of d-glucose uptake, indicating that it has a low affinity for the d-glucose transport system. The apparent K(m) of d-glucose transport was in the range of 5 to 15 mM, with a V(max) of 200 to 300 nmol min mg of protein. The K(m) of Z. mobilis glucokinase (0.25 to 0.4 mM) was 1 order of magnitude lower than the K(m) for d-glucose transport, although the V(max) values for transport and phosphorylation were similar. Thus, glucose transport cannot be expected to be rate limiting at concentrations of extracellular glucose normally used in fermentation processes, which greatly exceed the K(m) for the transport system. The low-affinity, high-velocity, nonconcentrative system for d-glucose transport described here is consistent with the natural occurrence of Z. mobilis in high-sugar environments and with the capacity of Z. mobilis for rapid conversion of glucose to metabolic products with low energetic yield.     

Mechanism and regulation of phosphate transport in Streptococcus pyogenes.

In contrast to results reported with other bacteria, uptake of 32Pi in Streptococcus pyogenes was found to occur rapidly in starved cultures and to be strongly and immediately inhibited by addition of exogenous glycolytic energy sources (such as glucose) and nonglycolytic sources of ATP (such as arginine). Preincubation of starved cells with NaF, iodoacetate, or arsenate eliminated the inhibiting effect of glucose but not that of arginine. In accordance with the hypothesis that transport was attributable to Pi-Pi exchange, uptake and efflux of 32Pi in the presence of trans unlabeled Pi exhibited similar characteristics and were largely eliminated by reduction of the trans Pi concentration. Neither process was inhibited appreciably by pretreatment of cells with ionophores or metabolic inhibitors, but both processes were abolished by exposure to p-chloromercuribenzoate. Inhibition by both exogenous energy sources resulted in a reduction in the maximal velocity of transport (Vmax). Whereas arginine also caused a shift in the apparent Michaelis-Menten constant (Km) to larger values, glucose did not alter the Km. On the basis of the results reported, we propose that the rate of Pi exchange is determined positively by the intracellular and extracellular concentrations of Pi and negatively by ATP or metabolites thereof. The mechanism of ATP action is unknown but could involve either covalent or noncovalent modification of the carrier protein.     

Effect of erythropoietin on the glucose transport of rat erythrocytes and bone marrow cells

The effect of Ep on radioactive glucose and methyl-alpha-D-glucoside transport by rat erythrocytes and bone marrow cells were studied. There is initial linearity followed by saturation kinetics of [14C]glucose transport by the erythrocytes of starved and starved plus Ep-treated rats at different concentrations of glucose. Starvation caused slight inhibition of glucose transport which increased markedly on Ep administration to starved rats. Normal animals failed to show any significant change in glucose transport after Ep treatment. Methyl-alpha-D-glucoside inhibited the Ep-stimulated glucose transport significantly. Ep also stimulated the transport of radioactive methyl-alpha-D-glucoside which was competitively inhibited in presence of D-glucose. Glucose transport in erythrocytes was found to be sensitive to metabolic inhibitors like azide and DNP. A sulfhydryl reagent and ouabain also inhibited the transport process. Ep stimulated glucose and methyl-alpha-D-glucoside transport in the bone marrow cells of starved rats. The sugar analog competitively inhibited the glucose transport in bone marrow cells and vice versa.     

A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression

Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3beta resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth.