Glucose transport into human erythrocytes treated with phospholipase A2 or C

Phospholipase A2 induced crenation of human erythrocytes and decreased glucose transport activity (influx rate) by 40% when 51% of phosphatidylcholine (PC) in the membrane was hydrolyzed. On the other hand, phospholipase C induced invagination of the cells and negligibly affected the glucose transport in the case of 21% hydrolysis of the PC. By altering the pH of the medium for suspending cells treated with phospholipase A2 from 7.4 to 6.0, cell shape was changed from clear crenation to slight invagination, but glucose transport activity was not affected. Cells that were treated with phospholipase A2 and then washed with albumin to remove free fatty acids produced in the cell membrane showed an almost normal cell shape and slightly higher glucose transport activity than did untreated cells. The ratios of beta-D-glucose transport rate to alpha-D-glucose transport rate in untreated cells, cells treated with phospholipase A2 and cells treated with phospholipase C were 1.13, 1.04, and 1.20, respectively. These results demonstrate that the drastic morphological change (invagination or crenation) induced by the treatment with phospholipases bears no clear relationship to the activity of glucose transport and suggest that the increase in the volume of the outer half of the lipid bilayer might reduce the rate of glucose transport across the human erythrocyte membrane and change the anomeric preference of glucose transport.     

Anomeric specificity of the stimulatory effect of D-glucose on D-fructose phosphorylation by human liver glucokinase

D-Glucose was recently reported to stimulate d-fructose phosphorylation by human B-cell glucokinase. The present study aims at investigating the anomeric specificity of such a positive cooperativity. The alpha-anomer of D-glucose was found to increase much more markedly than beta-D-glucose the phosphorylation of D-fructose by human liver glucokinase. Such an anomeric preference diminished at high concentrations of the D-glucose anomers, i.e. when the effect of the aldohexose upon d-fructose phosphorylation became progressively less marked. A comparison between the effects of the two anomers of D-glucose and those of equilibrated D-glucose upon D-fructose phosphorylation by human liver glucokinase indicated that the results obtained with the equilibrated aldohexose were not significantly different from those expected from the combined effects of each anomers of D-glucose. In isolated rat islets incubated for 60 min at 4 degrees C, alpha-D-glucose (5.6 mm), but not beta-D-glucose (also 5.6 mm), augmented significantly the conversion of D-[U-(14)C]fructose (5.0 mm) to acidic radioactive metabolites. Likewise, in islets prelabeled with (45)Ca and perifused at 37 degrees C, D-fructose (20.0 mm) augmented (45)Ca efflux and provoked a biphasic stimulation of insulin release from islets exposed to alpha-D-glucose (5.6 mm), while inhibiting (45)Ca efflux and causing only a sluggish and modest increase in insulin output from islets exposed to beta-D-glucose (also 5.6 mm). The enhancing action of D-glucose upon D-fructose phosphorylation by glucokinase thus displays an obvious anomeric preference for alpha-D-glucose, and such an anomeric specificity remains operative in intact pancreatic islets.     

Anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms

The anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms was investigated. For this purpose, the hemidiaphragms were preincubated for 30 min at 37 degrees C and then incubated for 5 min at the same temperature in the presence of alpha- or beta-D-[U-14C]glucose. The concentrations of D-glucose (5.6 or 8.8 mM) and insulin (0 or 10 mU/ml) were identical during the preincubation and incubation periods. The incubation medium was prepared in D2O/H2O (3:1, v/v) in order to delay the interconversion of the D-glucose anomers. In addition to glycogen labelling, the output of radioactive acidic metabolites was also measured. Insulin caused a preferential stimulation of glycogen labelling relative to glycolysis. Such was not the case in response to a rise in D-glucose concentration. At 5.6 mM D-glucose and whether in the presence or absence of insulin, both glycogen labelling and glycolysis were lower with alpha-D-glucose than with beta-D-glucose suggesting a higher rate of beta-D-glucose than alpha-D-glucose transport across the plasma membrane. A mirror image was found at 8.8 mM D-glucose, especially in the absence of insulin. At this close-to-physiological hexose concentration, insulin lowered the alpha/beta ratio for glycogen labelling. On the contrary, the rise in D-glucose concentration increased such a ratio. Since such a rise is probably little affected by any possible anomeric difference in D-glucose transport across the plasma membrane, the present results strongly suggest that the intracellular factors regulating net glycogen synthesis, as well as glycolytic flux, display obvious preference for alpha-D-glucose.     

Anomalous asymmetric kinetics of human red cell hexose transfer: role of cytosolic adenosine 5'-triphosphate

Cytosolic adenosine 5'-triphosphate (ATP) modifies the properties of human red cell sugar transport. This interaction has been examined by analysis of substrate-induced sugar transporter intrinsic fluorescence quenching and by determination of Michaelis and velocity constants for D-glucose transport in red cell ghosts and inside-out vesicles lacking and containing ATP. When excited at 295 nm, human erythrocyte ghosts stripped of peripheral proteins display an emission spectrum characterized by a scattering peak and a single emission peak centered at about 333 nm. Addition of sugar transport substrate or cytochalasin B and phloretin (sugar transport inhibitors) reduces emission peak height by 10% and 5%, respectively. Cytochalasin B induced quenching is a simple saturable phenomenon with an apparent Kd (app Kd) of 60 nM and a capacity of 1.4 nmol of sites/mg of membrane protein. Quenching by D-glucose (and other transported sugars) is characterized by at least two (high and low) app Kd parameters. Inhibitor studies indicate that these sites correspond to sugar efflux and influx sites, respectively, and that both sites can exist simultaneously. ATP induces quenching of stripped ghost fluorescence with half-maximal effects at 20-30 microM ATP. ATP reduces the low app Kd and increases the high app Kd for sugar-induced fluorescence quenching. D-Glucose transport in intact red cells is asymmetric (Km and Vmax for influx less than Km and Vmax for efflux). In addition, two operational Km parameters for efflux are detected in zero- and infinite-trans efflux conditions. Protein-mediated sugar transport in ghosts and inside-out vesicles (IOVs) is symmetric with respect to Km and Vmax for entry and exit, and only one Km for exit is detected. Addition of millimolar levels of ATP to the interior of ghosts or to the exterior of IOVs restores both transport asymmetry and two operational Km parameters for native efflux. A model for red cell hexose transport is proposed in which ATP modifies the catalytic properties of the transport system. This model mimics the behavior of the sugar transport systems of intact cells, ghosts, and inside-out vesicles.     

Inhibitions of sugar transport produced by ligands binding at opposite sides of the membrane. Evidence for simultaneous occupation of the carrier by maltose and cytochalasin B

This study examines inhibitions of human erythrocyte D-glucose uptake at ice temperature produced by maltose and cytochalasin B. Maltose inhibits sugar uptake by binding at or close to the sugar influx site. Maltose is thus a competitive inhibitor of sugar uptake. Cytochalasin B inhibits sugar transport by binding at or close to the sugar efflux site and thus acts as a noncompetitive inhibitor of sugar uptake. When maltose is present in the uptake medium, Ki(app) for cytochalasin B inhibition of sugar uptake increases in a hyperbolic manner with increasing maltose. When cytochalasin B is present in the uptake medium, Ki(app) for maltose inhibition of sugar uptake increases in a hyperbolic manner with increasing cytochalasin B. High concentrations of cytochalasin B do not reverse the competitive inhibition of D-glucose uptake by maltose. These data demonstrate that maltose and cytochalasin B binding sites coexist within the glucose transporter. These results are inconsistent with the simple, alternating conformer carrier model in which maltose and cytochalasin B binding sites correspond to sugar influx and sugar efflux sites, respectively. The data are also incompatible with a modified alternating conformer carrier model in which the cytochalasin B binding site overlaps with but does not correspond to the sugar efflux site. We show that a glucose transport mechanism in which sugar influx and sugar efflux sites exist simultaneously is consistent with these observations.     

Nicotinamide is not a substrate of the facilitative hexose transporter GLUT1

It has been proposed that GLUT1, a membrane protein that transports hexoses and the oxidized form of vitamin C, dehydroascorbic acid, is also a transporter of nicotinamide (Sofue, M., Yoshimura, Y., Nishida, M., and Kawada, J. (1992) Biochem. J. 288, 669-674). To ascertain this, we studied the transport of 2-deoxy-D-glucose, 3-O-methyl-D-glucose, and nicotinamide in human erythrocytes and right-side-out and inside-out erythrocyte membrane vesicles. The transport of nicotinamide was saturable, with a K(M) for influx and efflux of 6.1 and 6.2 mM, respectively. We found that transport of the hexoses was not competed by nicotinamide in both the erythrocytes and the erythrocyte vesicles. Likewise, the transport of nicotinamide was not affected by hexoses or by inhibitors of glucose transport such as cytochalasin B, genistein, and myricetin. On the other hand, nicotinamide blocked the binding of cytochalasin B to human erythrocyte membranes but did so in a noncompetitive manner. Using GLUT1-transfected CHO cells, we demonstrated that increased expression of GLUT1 was paralleled by a corresponding increase in hexose transport but that there were no changes in nicotinamide transport. Moreover, nicotinamide failed to affect the transport of hexoses in both control and GLUT1-transfected CHO cells. Therefore, our results indicates that GLUT1 does not transport nicotinamide, and we propose instead the existence of other systems for the translocation of nicotinamide across cell membranes.     

Erythrocyte D-glucose transport activity in reconstituted model membranes of different lipid composition

The stereospecific influx of D-glucose into liposomes formed on sonication of different glyco- and phospholipids with transport proteins from human erythrocyte ghosts solubilized with Triton x-100 was measured as an index of their total D-glucose transport activity. Specific D-glucose transport increased when acidic phospho- and glycolipids (especially sulfatide) were added to the phosphatidylcholine bilayers of the model membranes while cholesterol strongly inhibited the process. The modulation of D-glucose transport activity and its possible correlation with the lipid composition and the chemico-physical state of the erythrocytes is discussed.     

ATP regulation of the human red cell sugar transporter

Purified human red blood cell sugar transport protein intrinsic tryptophan fluorescence is quenched by D-glucose and 4,6-ethylidene glucose (sugars that bind to the transport), phloretin and cytochalasin B (transport inhibitors), and ATP. Cytochalasin B-induced quenching is a simple saturable phenomenon with Kd app of 0.15 microM and maximum capacity of 0.85 cytochalasin B binding sites per transporter. Sugar-induced quenching consists of two saturable components characterized by low and high Kd app binding parameters. These binding sites appear to correspond to influx and efflux transport sites, respectively, and coexist within the transporter molecule. ATP-induced quenching is also a simple saturable process with Kd app of 50 microM. Indirect estimates suggest that the ratio of ATP-binding sites per transporter is 0.87:1. ATP reduces the low Kd app and increases the high Kd app for sugar-induced fluorescence quenching. This effect is half-maximal at 45 microM ATP. ATP produces a 4-fold reduction in Km and 2.4-fold reduction in Vmax for cytochalasin B-inhibitable D-glucose efflux from inside-out red cell membrane vesicles (IOVs). This effect on transport is half-maximal at 45 microM ATP. AMP, ADP, alpha, beta-methyleneadenosine 5'-triphosphate, and beta, gamma-methyleneadenosine 5'-triphosphate at 1 mM are without effect on efflux of D-glucose from IOVs. ATP modulation of Km for D-glucose efflux from IOVs is immediate in onset and recovery. ATP inhibition of Vmax for D-glucose exit is complete within 5-15 min and is only partly reversed following 30-min incubation in ATP-free medium. These findings suggest that the human red cell sugar transport protein contains a nucleotide-binding site(s) through which ATP modifies the catalytic properties of the transporter.     

Organic-acid transport in resealed haemoglobin-containing human erythrocyte ''ghosts''.

The transport of organic acids across the membrane of resealed haemoglobin-containing erythrocyte 'ghosts' prepared by a dialysis technique has been studied. The present work forms part of studies directed towards the use of erythrocyte cellular carriers in enzyme-replacement therapy of inherited metabolic diseases. Oxalic acid, glycollic acid and glyoxylic acid were taken as representative of aliphatic acids of low molecular mass and benzoic and cinnamic acids as representative of unsubstituted aromatic acids. These selected acids are important in the diseases with which the present work is concerned. Comparison of influx and efflux transport characteristics showed that erythrocyte 'ghosts' retain transport properties closely similar to those of normal erythrocytes. Rapid transport was observed with all organic acids studied and there was a linear relationship between initial amount of influx and external concentration of aliphatic acid. Saturation of the transport system was not observed up to 1 mM external concentration, and the presence of plasma in the external medium had no effect on transport characteristics. Transport in intact erythrocytes and prepared erythrocyte 'ghosts' from patients with hyperoxaluria was also studied.     

Electrical properties and glucose permeability of bilayer lipid membranes on incorporation of erythrocyte membrane extracts

1. Extracts of the human erythrocyte membrane have been prepared by solubilization with Triton X-100 and analysed by electrophoresis and gel filtration techniques. 2. The extracts have been incorporated asymmetrically into lecithincholesterol-n-decane planar bilayers. 3. The electrical characteristics and glucose permeabilities of the bilayers have been measured. 4. The extracts increased the electrical conductance of the bilayers and also markedly enhanced the D-glucose permeability but not the L-glucose permeability. 5. The enhanced D-glucose permeability was inhibited by monosaccharide transport inhibitors. 6. The results support the claim that a monosachharide facititated diffusion system has been set-up in vitro which has many of the characteristics of the transport system in the human erythrocyte membrane. 7. The data indicates that the trans membrane polypeptides of band 3 of the electrophoretogram of the erythrocyte membrane proteins (notation of Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2616) are implicated in D-glucose transport, although the possibility that relatively minor component of the membrane could be responsible for glucose transport cannot be eliminated.     

Sugar transport asymmetry in human erythrocytes--the effect of bulk haemoglobin removal and the addition of methylxanthines

1. The glucose transport asymmetry of intact human red cells has been shown to be retained in pink erythrocyte ghosts (a preparation of membranes in which 95% of the red cell haemoglobin has been removed). 2. 3-Isobutyl-1-methylxanthine inhibits net glucose efflux in intact cells and ghosts and also net influx in cells. 5mM theophylline inhibits net efflux in ghosts. The inhibition type is mixed. The major effect is a decrease in the V value for net flux but a small increase in Km also occurs. 3-Isobutyl-1-methylxanthine binds the transport system from the external solution only. 3. Exchange flux of glucose shows virtually no inhibition by 3-isobutyl-1-methylxanthine. 4. The results are discussed in terms of models for sugar transport. A model consistent with the observed pattern of inhibition would be one in which transport is rate-limited by the membrane and in which net and exchange flux occur via separate transport cycles.     

Effect of magnesium-dependent cell membrane alterations on the transport of K+ in Ehrlich ascites tumour cells

Mg-deficiency or Mg-loading of tumour cells changes the permeability of the cell membrane. The influence of this change on the K+ transport across the membrane was investigated using 86Rb+ and K+ analog. The time course of the influx and efflux rates were estimated by means of a mathematical approach for a two-compartment system with inconstant pool sizes. The comparison of the two states of the cells demonstrates that in Mg-deficient cells the passive K+ efflux is significantly enhanced (40%). This in turn stimulates the active counter transport mediated by the (Na+-K+)-ATPase, raising the ATP consumption by about 30%. However, the enzyme is not able to maintain the cellular K+ content under these conditions. After a short transient increase due to the initially enhanced influx the passive net efflux prevails. Differences in the electrophoretic mobility of the two states of the cells confirm Mg-dependent changes of the cell membrane structure.     

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity

We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux.     

Biochemical studies of the excitable membrane of Paramecium aurelia. I. 45Ca2+ fluxes across resting and excited membrane.

The characteristics of Ca2+ transport across the excitable membrane of Paramecium aurelia were studied by measuring 45Ca2+ influx and efflux. The intracellular concentration of free Ca2+ in resting P. aurelia was at least ten times less than the extracellular concentration. Ca2+ influx was easily measurable at 0 degrees C, but not at 23 degrees C. The influx of 45Ca2+ was stimulated by the same conditions which cause membrane depolarization and ciliary reversal. Addition of Na+ and K+ (which stimulate ciliary reversal) resulted in a 10-fold increase in the rate of Ca2+ influx. An externally applied, pulsed, electric field (1-2 mA/cm2 of electrode surface), caused the rate of Ca2+ influx to increase 3-5 times, with the extent of stimulation dependent on the current density and the pulse width. Ca2+ influx had the characteristics of a passive transport system and was associated with the chemically or electrically triggered Ca2+ "gating" mechanism, which has been studied electrophysiologically. In contrast, Ca2+ efflux appeared to be catalyzed by an active transport system. With cells previously loaded at 0 degrees C with 45Ca2+, Ca2+ efflux was rapid at 23 degrees C, but did not occur at 0 degrees C. This active Ca2+ efflux mechanism is probably responsible for maintaining the low internal Ca2+ levels in unstimulated cells.     

The effect of the strongly bound protein fraction on sugar transport in human erythrocyte ghosts

The protein fraction released from human erythrocyte membranes with 90% acetic acid enhanced the transport of several sugar species when enclosed in erythrocyte ghosts. Both the influx and the efflux of d-glucose were increased so that permeation rather than sugar binding was involved. The permeation increase was selective, being found with d-glucose, d-galactose and d-xylose but not with l-glucose or lactose. The protein-dependent sugar transport was saturable and the incorporation of proteins into the ghost membrane brought J_max to the level corresponding to intact erythrocytes, leaving K_m unchanged.     

A rapid method of reconstituting human erythrocyte sugar transport proteins

A rapid reconstitution procedure for human erythrocyte hexose transfer activity is described. The procedure (reverse-phase evaporation) avoids exposure of the isolated proteins to detergent, organic solvent, sonication, or freeze-thaw steps during insertion into synthetic membranes and may be effected within 15 min. The so-formed vesicles are unilamellar structures with a large encapsulated volume, narrow size range, and low passive permeabilities. Contamination by carry-through of endogenous (red cell) lipids is less than 1%. Reconstituted hexose transfer activity was examined by using unfractionated proteins (bands 3, 4.5, and 6) and purified proteins (bands 4.5 and 3). With unfractionated proteins, hexose transport activity is low [0.34 mumol X (mg of protein)-1 X min-1], is inhibited by cytochalasin B, and increases monotonically with protein concentration. Kinetic analysis indicates that Vmax values for both influx and efflux of D-glucose are identical. Reconstitution of the cytochalasin B binding protein (band 4.5) results in hexose transport with high specific activity [5 mumol X (mg of protein)-1 X min-1] and symmetry in transfer kinetics. Band 3 proteins also appear to mediate cytochalasin B sensitive D-glucose transport activity.     

Distinction of three types of D-glucose transport systems in animal cells

Immunoblotting of plasma membrane fractions from rat kidney cortex with antibody to human erythrocyte glucose transporter showed a single major cross-reacting material of 48K in basolateral membrane fractions possessing a facilitated diffusion system for D-glucose, but not in brush border membrane fractions which have a Na-dependent active transport system. Cytochalasin B inhibited D-glucose uptake in basolateral membrane vesicles but not in brush border vesicles. Cross-reacting materials of 44-55K were detected in several animal cells exhibiting facilitated diffusion systems, including a hormone dependent system. These results indicate molecular difference between glucose transporters of facilitated diffusion systems and active transport systems.     

D-glucose anomeric preference of hexokinases from animals and yeast

The D-glucose anomeric preference of hexokinases partially purified from animals (rat, mouse, and chicken) and baker's yeast (Saccharomyces cerevisiae) were investigated by the assay system with glucose-6-phosphate dehydrogenase as a coupling enzyme. With low Km hexokinases in animal tissues and cells, the ratios of Vmax for the beta-anomer to Vmax for the alpha-anomer (V beta/V alpha) were within a range from 1.3 to 1.5. In yeast, the V beta/V alpha value was 1.1 for hexokinase A, 0.8 for hexokinase B, and 1.4 for glucokinase. The possible explanation for D-glucose anomeric preference of hexokinase is discussed.     

Reconstitution of glucose-transporting vesicles from erythrocyte membranes disaggregated in detergent.

With the eventual aim of purifying a membrane transport system by using reconstitution of transport activity as an assay, I showed that if, after the erythrocyte membrane is solubilized in deoxycholate, the detergent is removed, membrane vesicles re-form which retain glucose-transport activity. They take up and release D-glucose in preference to L-glucose and the uptake and release are sensitive to Hg2+ and phloretin. Release of tracer D-glucose is competitively inhibited by transported sugars inside the vesicles and increased by unlabelling D-glucose in the outside medium. Uptake of tracer is increased so much by preloading vesicles with unlabelled transported sugars that the tracer is probably concentrated against a gradient. When the membrane is solubilized, two proteins that span the membrane can be separated, suggesting that it will be possible to fractionate the membrane before reconstitution.     

Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.