Human Red Cell Glucose Transporter Research Articles

Ethanol weakens cytochalasin B binding to the GLUT1 glucose transporter and drug partitioning into lipid bilayers

Ethanol weakens the specific interaction between the human red blood cell (RBC) glucose transporter GLUT1 and the inhibitor cytochalasin B (CB). The chromatographic retention volume of cytochalasin B on stationary phases consisting of GLUT1-containing membranes decreased with increasing ethanol concentration in the eluent. The apparent K d values for the ethanol–GLUT1 interaction were 0.37, 0.45 and 0.64 M for red blood cells, red blood cell membrane vesicles and proteoliposomes, respectively, all much higher than the K d values for d-glucose or cytochalasin B interaction with GLUT1. Ethanol also decreased the partitioning of cytochalasin B and drugs into phospholipid bilayers.

Chromatography on cells: analyses of solute interactions with the glucose transporter Glut1 in human red cells adsorbed on lectin-gel beads

The affinities of the human red cell glucose transporter Glut1 for D-glucose and cytochalasin B (CB) and the stoichiometry of CB binding vary with the Glut1 environment. In order to study the native state of Glut1 we adsorbed human red cells to wheat germ lectin agarose gel beads for frontal affinity chromatographic analyses. Glut1 showed relatively high affinities for D-glucose ( K d 12±1 m M) and CB ( K d 59±17 n M). The number of CB-binding sites per Glut1 monomer, 0.46±0.16, was approximately doubled upon coating the cells with polylysine, which induced cell association.

Biomembrane-affinity centrifugal analyses of solute interactions with membrane proteins

We have developed a rapid centrifugal method for analyzing solute interactions with membrane proteins in cytoskeleton-depleted membrane vesicles or proteoliposomes sterically immobilized in Superdex 200 gel beads. The size and density of the gel beads allow fast sedimentation in a bench-top centrifuge. Biospecific interactions of cytochalasin B and d-glucose with the human red cell glucose transporter, Glut1, were analyzed. The binding constants and the molar ratio of inhibitor sites per protein monomer agreed well with recent results obtained by frontal affinity chromatography.

Biomembrane affinity chromatographic analysis of nitrobenzylthioinosine binding to the reconstituted human red cell nucleoside transporter.

Solute interactions with membrane proteins can be analyzed by biomembrane affinity chromatography (BAC), previously applied to the human red cell glucose transporter. As a novel example, frontal BAC analysis of interactions between the nucleoside transport inhibitor nitrobenzylthioinosine (NBTI) and immobilized reconstituted nucleoside and glucose transporters from human red cells revealed two binding sites, presumably corresponding to the two transporters. The affinities and amounts of sites were determined by use of a double rectangular hyperbolic equation. The Kd value for NBTI binding to the nucleoside transporter in egg phospholipid proteoliposomes was 0.38 +/- 0.08 nM (22 degrees C, I = 0.16, pH 7.4), lower than previously reported for reconstituted systems. The molar ratio between the amounts of nucleoside transporter sites for NBTI and glucose transporter sites for cytochalasin B was 4.5 +/- 0.6%.

D-Glucose, forskolin and cytochalasin B affinities for the glucose transporter Glut1: Study of pH and reconstitution effects by biomembrane affinity chromatography

The affinities of d-glucose and the transport inhibitors, forskolin and cytochalasin B (CB), for Glut1 were studied by frontal affinity chromatography at pH 5–10 on sterically immobilized proteoliposomes with reconstituted human red cell glucose transporter Glut1. The affinity of d-glucose for Glut1 became slightly weaker as the pH was increased. The inhibitor affinities decreased and became immeasurably weak above pH 9. At pH 7.4, the dissociation constants were 44 mM for glucose, 1.8 μM for forskolin and 72 nM for CB. The affinities of these solutes for Glut1 in red cell membrane vesicles and particularly for Glut1 in red cells were higher, as shown by chromatographic analyses.

Frontal affinity chromatographic analysis of membrane protein reconstitution

The human red cell glucose transporter Glut1 was solubilized with octaoxyethylene n-dodecyl ether (low critical micelle concentration (CMC)), purified, mixed with egg phospholipids and cholate, and reconstituted by gel filtration on Superdex 75. Free protepliposomes showed relatively high D-glucose transport activity. Frontal affinity chromatographic analysis with the proteoliposomes sterically immobilized in Superdex 200 gel beads revealed that the number of operative cytochalasin B (CB) binding sites increased during the first days of chromatographic runs to become the same as with 1- O- n-octyl β- d-glucopyranoside (high CMC) as solubilizer and Sephadex G-50 as gel filtration medium. The average number of sites per Glut1 monomer was 0.32 ± 0.02. The average K d for CB was 66 ± 3 nM at 150 mM NaCl, similarly as for Glut1 in membrane vesicles, whereas the affinity of d-glucose for reconstituted Glut1 was lower ( K d = 44 ± 3 mM) than for membranous Glut1 ( K d = 15 ± 5 mM). Two theoretical treatments of affinity chromatographic data gave the same values in agreement with competitive and monovalent interactions.

Monomeric human red cell glucose transporter (Glut1) in non-ionic detergent solution and a semi-elliptical torus model for detergent binding to membrane proteins

The self-association state of the human red cell glucose transporter (Glut1) in octaethylene glycol n-dodecyl ether (C 12E 8) and n-octyl β- d-glucopyranoside (OG) solution was analyzed in the presence of reductant by gel filtration with light-scattering, refractivity and absorbance detection, and by ultracentrifugation. The C 12E 8-Glut1 complex was essentially monomeric, whereas OG-Glut1 also formed dimers and larger oligomers. C 12E 8-Glut1 retained substantial glucose transport activity even after depletion of endogenous lipids by gel filtration, as shown by reconstitution and transport measurements. Removal of endogenous lipids from OG-Glut1 abolished the activity unless phosphatidylcholine was included in the eluent. The binding of C 12E 8 and OG to Glut1 was determined by gel filtration with refractivity and absorbance detection or with radioactive tracer to be 1.86 ± 0.07 and 1.84 ± 0.09 g/g polypeptide, respectively. A structural model was proposed in which non-ionic detergent forms a semi-elliptical torus (SET) surrounding the transmembrane protein. The torus thickness was assumed to be equal to the radius (short half-axis) of a spherical (oblate ellipsoidal) free detergent micelle and the polar head groups of the detergent molecules were predicted to be situated just outside the hydrophobic surface of the protein. The experimental detergent binding values and those obtained from the SET model together confirmed that Glut1 was monomeric in C 12E 8 solution and provided constraints on the shape and size of the hydrophobic transmembrane region of Glut1 in α-helical and β-barrel topology models.

Capillary and rotating-tube isoelectric focusing of a transmembrane protein, the human red cell glucose transporter.

The human red cell glucose transporter (Glut1) is a transmembrane protein. Monomeric Glut1 was purified by ion-exchange chromatography in the presence of the non-ionic detergent n-dodecyl octaoxyethylene (C12E8). For focusing, the ionic strength of the solution of C12E8-Glut1 complexes with co-purified lipids was lowered by dialysis, the detergent concentration was increased and carrier ampholytes were added. Focusing was done for 5 min at 3000 V in a methyl cellulose-coated glass capillary (50 microns I.D.). The anolyte H3PO4 was then replaced by NaOH for mobilization towards the anode. Absorbance monitoring at 280 nm showed two groups of zones at pH 6 and 8. Similarly, isoelectric focusing in a rotating quartz tube (3 mm I.D.) gave Glut1 zones at pH 5.5 and 8.0. Phosphorus analysis revealed that the Glut1 zone at pH 8 contained more phospholipids than did the other one. The above results together with previously determined and calculated isoelectric points (pI) of Glut1 indicate that the Glut1 at pH 8 is monomeric and that the zone at pH 5.5-6 represents oligomeric materials. The pI 8.0 at 22 degrees C applies for monomeric Glut1 in the absence of urea. The results exemplify that capillary isoelectric focusing of hydrophobic membrane proteins is possible.

Immobilized Liposome Chromatography for Analysis of Interactions Between Lipid Bilayers and Peptides

Liposomes were sterically immobilized in gel beads to be used for isocratic chromatographic analysis of interactions between lipid bilayers, amino acids, and water-soluble peptides. Tryptophan was more retarded on the immobilized liposomes than were other amino acids, and peptides with C-terminal cysteine were much more retarded than were peptides with serine or aminobutyric acid instead of the cysteine. Peptide sequences could affect the peptide-liposome interactions. For peptides corresponding to polypeptide segments of a membrane protein, the human red cell glucose transporter (Glut1), the retention volumes increased with decreasing water-to-oil transfer free energy of the peptides and were related to the transfer free energy distribution within the peptides as shown by hydropathy plots and to the presence of cysteine. Among these peptides, the partially hydrophobic peptides 125GRFIIGVYCG 134 and 201CIVLPFCPES 210 and the hydrophilic peptide 421CFQYVEQLC 429 were most strongly retarded on immobilized phosphatidylcholine liposomes. The results indicated that the peptide-liposome interactions were mainly of hydrophobic nature and that the structure of the interfacial headgroup region of the lipid bilayers was important.

Immobilized proteoliposome affinity chromatography for quantitative analysis of specific interactions between solutes and membrane proteins. Interaction of cytochalasin B and D-glucose with the glucose transporter Glut1.

An affinity gel bed was prepared by reconstitution of a transmembrane protein, the human red cell glucose transporter (Glut1), followed by steric immobilization of the proteoliposomes in small and rigid gel beads by freeze-thawing. The specific interactions between the reconstituted Glut1, the transport inhibitor cytochalasin B (CB), and the transported solute D-glucose were analyzed by isocratic chromatography of CB on the Glut1-proteoliposome gel bed. Specific retardation of CB which decreased upon inclusion of the competitor D-glucose in the eluent was observed on-line. The equilibrium constants for CB and D-glucose interaction with Glut1 (Kd 1.5 x 10(-7) M and 67 mM, respectively) obtained by use of equations derived for the affinity chromatographic analysis were consistent with values obtained by others by conventional methods. Effects of liposome composition, pH, and time on the CB binding activity of Glut1 were studied. Reconstitution of a membrane protein into a lipid environment and steric immobilization of the proteoliposomes favor retention of the protein activity. Immobilized proteoliposome affinity chromatography (IPAC) is a novel, powerful method for analysis of interactions between membrane proteins and solutes.

Calculation of the isoelectric points of native proteins with spreading of pKa values.

The isoelectric points (pI) of native proteins are important in several separation techniques. For estimating pI values the net charge of several proteins was calculated versus pH by use of the Henderson-Hasselbalch equation. Amino acid composition, pKa values for amino acid side chains and for the N- and C-terminal groups, and the presence of other charged groups were taken into account. A set of pKa values was chosen for amino acid residues with ionizable side chains. Each particular type of ionizable group was assumed to have pKa values distributed around the chosen value, thereby simulating the situation in proteins and polypeptides. The calculated pI values showed reasonably good agreement with experimental ones for most of 16 native proteins over a wide pH range (3.4-11) when charge contributions of heme groups, sialic acid residues, etc., were taken into account. The calculated pI for the human red cell glucose transporter (Glut1) with one sialic acid residue was decreased from 8.8 to 8.5 by introducing pKa value spreading and became consistent with the experimental pI value of 8.4 +/- 0.05 at 15 degrees C determined in the presence of 6 M urea. The pI of the native Glut1 was lower, 8.0 +/- 0.1, at 22 degrees C. In general, the pI values for native proteins are affected by the three-dimensional structure of the proteins, which causes greater differences between calculated and experimental pI values than in the case of polypeptides for which pI values are determined in the presence of urea.

Effects of pH on the activity of the human red cell glucose transporter Glut 1: transport retention chromatography of d-glucose and l-glucose on immobilized Glut 1 liposomes

The facilitative glucose transporter Glut 1 from human red cells was reconstituted into liposomes that were size-fractionated and immobilized in an octyl sulfide-Sephacryl S-1000 column. d-[ 14C]Glucose was eluted later than l-[ 3H]glucose from the Glut 1 liposome column ( by δV μ l ), apparently because the d-glucose was transported through the liposomes. The corresponding difference with protein-free liposomes was δV 0 . The Glut 1 transport retention chromatographic effect, δV G = δV − δV 0 , 40–50 μl at pH 7, was nearly constant at pH 6–10 ( 400 mM NaCl, 23° C, internal liposome volume ≈ 240μ l ) but decreased steeply below pH 5 to become zero at pH 3.6. The decrease corresponded to a pK a of ≈ 4.4 and was partly reversible above pH 4.7. Similarly, glucose exchange by non-immobilized freeze-thawed proteoliposomes with Glut 1 slowed down drastically as the pH was lowered from pH 5.5 to 4; and octyl glucoside-solubilized Glut 1 lost half its activity in 15 min at pH 4.5 (low ionic strength, 2°C) as shown by glucose exchange determinations at pH 7.2. The results suggest that Glut 1 is inactivated at low pH upon protonation of carboxylate groups of pK a ≈ 4.4–4.8 . It seems likely that carboxylate groups form hydrogen bonds to transported d-glucose.

Isoelectric focusing in immobilized pH gradients of the glucose transporter from human red cell membranes.

Isoelectric focusing of the human red cell glucose transporter (a transmembrane protein) was performed in immobilized pH gradients. Isoelectric focusing of integral membrane proteins presents problems that are related to the amphiphilic nature of these proteins. Solubilizing additives must be used to counteract hydrophobic effects. In our case, urea and the nonionic detergent, Triton X-100, were used. Focusing was done at 15 degrees C. The isoelectric point (pI) of the glucose transporter (freshly purified by anion-exchange chromatography in the presence of octyl glucoside) was determined at 8.4 +/- 0.05 (n = 9), in good agreement with our earlier determinations by two-dimensional electrophoresis with isoelectric focusing in the presence of carrier ampholytes in the first dimension. The width of the focused zone was approximately 0.1 pH unit, more narrow than after focusing with carrier ampholytes. In an immobilized pH gradient from pH 7 to 10, the transporter region at pH 8.4 comprised one major and one or two minor zones. The pH interval 4-10 was also used and showed a single transporter zone. The glucose transporter tends to self-associate in detergent solution. Octyl glucoside-purified glucose transporter formed oligomers during incubation at 37 degrees C. Upon focusing, these oligomers appeared in a wide pH interval far below pH 8.4.

Effects of pressure on glucose transport in human erythrocytes

The operation of the human red cell glucose transporter has been studied at normal and high hydrostatic pressure to identify the step(s) which involve a volume change. Pressure inhibited zero- trans and equilibrium exchange influx to similar extents, by decreasing the V max but not significantly changing the K m. The B max and K d of specific [ 3H]cytochalasin B binding were unaffected by pressure indicating no change to the number or affinity of functional transporters at pressure. Passive glucose transport was inhibited by pressure in a manner consistent with permeation across the lipid bilayer. These data indicate that there is a major change in volume during the translocation step of the glucose transporter which is rate-limiting for transport.

Active and monomeric human red cell glucose transporter after high performance molecular-sieve chromatography in the presence of octyl glucoside and phosphatidylserine or phosphatidylcholine

The human red cell glucose transporter (Glut 1) was purified by ion-exchange chromatography in the presence of octyl glucoside. The state of association of the protein was studied, and the transport activity was determined after exchange of copurified membrane lipids for phosphatidylserine (PS) or phosphatidylcholine (PC). The purpose was to analyze the Glut 1 preparation for homogeneity and activity prior to attempts at crystallization. Analyses by high performance molecular-sieve chromatography showed that the Glut 1 was monomeric immediately after the ion-exchange purification: the M r of the Glut 1 polypeptide was estimated to be 49 000 ± 6000 by TSKgel G3000SW chromatography monitored by low-angle laser light-scattering photometry, differential refractometry and UV photometry. This required determination of the absorption coefficient of the Glut 1, which was measured to be 1.13 ± 0.03ml mg −1 cm −1 at 280 nm, referring to the polypeptide concentration. The M r value is consistent with the cDNA-deduced M r 54117 of the very similar HepG2 glucose transporter polypeptide. At 2°C, pH 7 and an ionic strength of 0.06 M, the Glut 1 associated gradually during three days to form oligomers. These formed much more rapidly at room temperature or at high ionic strength. Freshly prepared Glut 1 retained high activity after separation from membrane lipids on a TSKgel G3000SW column in the presence of 40 mM octyl glucoside and 1 mM PS or PC. In contrast, most of the activity was lost when the membrane lipids were separated from the protein in the absence of eluent lipids. The presence of a phospholipid was thus essential for retention of high activity of the Glut 1 in octyl glucoside and PC was nearly as effective as PS.

The isoelectric point of the human red cell glucose transporter

The isoelectric point (p I) of human red cell glucose transporter (Glut 1) was determined. Inconsistent values of 6.0, 6.4–6.5 and 8 have been reported earlier. Integral membrane proteins from human red cells were analyzed by two-dimensional electrophoresis with isoelectric focusing and sodium dodecyl sulfate gel electrophoresis (2D-PAGE). A zone of monomeric Glut 1 was found at pH 8.7, but most of the Glut 1 focused at pH 6–7 together with the anion transporter and other components. Purified Glut 1 focused only at pH 8.5 ± 0.2 (S.D., n = 12) and deglycesylated purified Glut 1 only at pH 8.4 ± 0.1 ( n = 5), as shown by 2D-PAGE. The absence of Glut 1 below pH 8 in the latter cases was confirmed by immunoblotting with a monoclonal antibody. Furthermore, Glut 1 was photoaffinity-labelled with [ 3H]cytochalasin B and subjected to isoelectric focusing in one dimension. The p I of the labelled Glut 1 was 8.6 ± 0.3 ( n = 11). A p I of 9.1 was calculated for the Glut 1 polypeptide on the basis of amino acid composition and p K a values for amino acid side groups. The sialic acid content of the glycosylated transporter from fresh red cells was determined at approximately 2.1 sialic acid residues per transporter, which corresponds to a calculated p I of 8.8. The p I values of other human glucose transporter polypeptides of the facilitative diffusion type (Glut 2, 3, 4 and 5) were calculated at 8.4, 7.4, 7.1 and 6.2, respectively.

Substantial glucose leakage from liposomes on filters and upon molecular-sieve chromatography in determinations of reconstituted glucose-transport activity and liposome volumes

Transport-protein activities are often determined by procedures that involve isolation of liposomes containing the transported radioactive solute. We determined the activity of the human red cell glucose transporter in liposomes and, by similar procedures, internal volumes of liposomes. For these purposes, we isolated freeze-thawed liposomes loaded with [ 14C]glucose, either by filtration on cellulose-nitrate and cellulose-acetate filters, or by chromatography on Sephadex. The interaction of liposomes with filters caused substantial leakage of [ 14C]glucose. About half of the internal [ 14C]glucose was released on the filters from glucose-transporter liposomes with inhibited transport. Chromatography at high flow rate provided higher and more accurate values than did the filtration procedure. Leakage corrections could be made by use of flow-cell scintillation elution profiles. The ratios between the corrected chromatographic volume values and the filtration values were 1.4–3.0 for liposomes without protein, 2.4–4.0 for glucose-transporter liposomes and 3.6–7.9 for liposomes with several human red cell integral membrane proteins. The d-glucose equilibrium exchange with glucose-transporter liposomes at 50 mM d-glucose was 2.0 nmol d-glucose per μg transporter per second as determined by use of chromatography at high flow rate. The filtration procedure gave only 0.6 nmol · μg −1 · s −1 due to the [ 14C]glucose leakage. In our experiments, the chromatographic procedure thus proved superior.

Brain-type glucose transporter (GLUT-1) is selectively localized to the blood-brain barrier. Studies with quantitative western blotting and in situ hybridization.

The hypothesis that the GLUT-1 glucose transporter isoform is expressed selectively in brain at the capillary endothelium, i.e. the blood-brain barrier (BBB), was tested by using quantitative Western blotting, cytochalasin B binding, and in situ hybridization in bovine brain cortex. Purified human red cell glucose transporter was used as the standard for quantitative Western blots, because the mobility of the human erythrocyte and BBB glucose transporters in electrophoretic gels was identical. The concentration of immunoreactive glucose transporter in bovine BBB plasma membranes was 10.8 +/- 0.9 pmol/mgp (mean +/- S.E., n = 6). This value was not statistically different from the estimate of the maximal binding sites of D-glucose-displaceable [3H]cytochalasin B binding in the BBB membrane preparations, 11.7 +/- 3.5 pmol/mgp. In situ hybridization experiments using 35S-labeled antisense and sense riboprobes corresponding to nucleotides 385-932 of the GLUT-1 cDNA showed prominent hybridization of the antisense probe over brain microvascular endothelium, but no hybridization over neuropil greater than that found with the 35S-labeled sense probe. These studies are consistent with the following conclusion: (a) essentially 100% of the glucose transporter binding sites at the BBB can be accounted for by the GLUT-1 isoform; (b) in situ hybridization studies confirm previous Northern blot analysis and indicate the GLUT-1 gene is expressed selectively in microvascular endothelium in brain with minimal, if any, expression of this gene in neurons or glial cells in vivo.

Binding of sodium dodecyl suplhate to an integral membrane protein and to a water-soluble enzyme : Determination by molecular-sieve chromatography with flow scintillation detection

We have determined the binding of sodium dodecyl (SDS) to the human red cell glucose transporter (polypeptide, M r 54 117) and to a water-soluble enzyme, N-5′-phosphoribosylanthranilate isomerase—indole-3-glycerol-phosphate synthase (PRAI—IGPS) from Escherichia coli ( M r 49 484). [ 35S]SDS was equilibrated with each protein on molecular-sieve chromatography at a series of SDS concentrations. The binding ratios of SDS to protein were determined by flow scintillationdetection and automated amino acid analyses. Unexpectedly the glucose transp an automated amino acid analyses. Unexpectedly the glucose transporter, which is a transmembrane protein, bound about the same amount of SDS per gram of protein as did the enzyme. At 1.6 m M SDS, slightly below the critical micelle concentration (CMC) (1.8 m M) in the eluent, the binding ratio was 1.6 g SDS/G protein for both the glucose transporter and PRAI—IGPS. At 2.0 m M SDS (above the CMC) the glucose transporter showed a binding ratio of 1.7 g SDS/g protein. The corresponding value for the enzyme was about 1.5 g/g. The SDS—glucose transporter complex seems to be more compact than the SDS—enzyme complex as judged by molecular-sieve chromatography and by SDS—polyacrylamide gel electrophoresis. Tecent neutron scattering results have shown a protein-decorated triple-micelle struct scattering results have shown a protein-decorated triple-micelle structure for the SDS—PRAI—IGPS complex. Hypothetically, the more compact SDS—glucose transporter complex may therefore consist of a dual-micelle structure. The molecular-sieve gel beads bound considerable amounts of SDS. The SDS binding to gel matrix and to the proteins increased with increasing SDS concentration up to at least 1.6–2.0 m M SDS. In the case of the water-soluble enzyme a shoulder was observed in the binding curve at 1 m M SDS, probably reflecting a change in the conformation of the complex.

The human red cell glucose transporter in octyl glucoside. High specific activity of monomers in the presence of membrane lipids

Human red cell membranes were stripped of peripheral proteins and partially solubilized with 50–260 mM octyl glucoside at 2–14 mg protein/ml, to find conditions that afford a high concentration of active glucose transporter after purification on DEAE-cellulose. Transporter-egg yolk phospholipid vesicles were prepared by gel filtration. The specific d-glucose equilibrium exchange activities increased with increasing dilution of the glucose transporter. At 260 mM octyl glucoside the glucose transporter became partially denaturated. At 225 mM detergent the DEAE-cellulose chromatography showed one main and one minor fraction of active glucose transporter. Nucleoside transport activity was enriched in the minor fraction. Solubilization with 75 mM octyl glucoside at 8 mg protein/ml gave a maximal concentration of purified transporter, 0.8 mg/ml, probably corresponding to complete solubilization. The phospholipids were partially retarded on the DEAE-cellulose. The specific d-glucose equilibrium exchange was high, up to 200 nmol glucose / μg transporter in two min at 50 mM glucose. High performance gel filtration in octyl glucoside indicated that the transporter formed dimers during the fractionation. These eluted at M r 125 000, partially separated from the phospholipids, which appeared at M r 55 000 (cf. Mascher, E. and Lundahl, P. (1987) J. Chromatogr. 397, 175–186). The d-glucose transport activity was low in the main fraction and high in the transporter-phospholipid fraction. Mixing of these fractions did not increase the activity. The glucose transporter is probably dependent on one or more specific membrane lipid(s). Presumably the transporter dimerizes and loses activity upon removal of these lipids.