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/micrograms 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 Mr 125,000, partially separated from the phospholipids, which appeared at Mr 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.     

Identification of the d-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[³H]Cytochalasin B binding and its competitive inhibition by d-glucose have been used to identify the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [³H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for d-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the d-glucose-inhibitable site. If 280 nM (40 000 μunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of d-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Molecular characteristics of rat brain glucose transporter: a novel species with Mr 45,000

The glucose transporter of rat brain was examined by the use of cytochalasin B, a potent inhibitor. The dissociation constants (Kd) of D-glucose-inhibitable cytochalasin B binding in various membrane fractions were about 100 nM. Solubilization and partial purification of glucose transporter were carried out by procedures of DE 52 column chromatography, Bio Gel HT column chromatography and Sepharose CL-6B column chromatography from postnuclear membrane fraction. Purified transporter, reconstituted in lipid vesicles, showed D-glucose-specific transport activity with a Michaelis constant (Km) of 7 mM. The molecular weight was estimated to be about 200K by gel filtration in the presence of 0.1% Triton X-100. The subunit molecular weight was estimated to be 45K by SDS-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a covalent probe, indicating that rat brain glucose transporter is a tetramer.     

Chromatographic characterization of nitrobenzylthioinosine binding proteins in band 4.5 of human erythrocytes: purification of a 40 kDa truncated nucleoside transporter

DEAE-column-purified band 4.5 polypeptides of human erythrocyte membranes are mostly glucose transporters with nucleoside transporters as a minor component. The purpose of the present work was to differentially identify and isolate the nucleoside transporters in band 4.5 free from glucose transporters. Equilibrium binding studies demonstrated that the band 4.5 preparation binds nibrobenzylthioinosine (NBTI), a potent nucleoside transport inhibitor, at two distinct sites, one with a high affinity (dissociation constant, KD of 1 nM) with a small capacity, BT (0.4 nmol/mg protein), and the other with a low affinity (KD of 15 microM) with a large BT (14-16 nmol/mg protein). The BT of the low-affinity site was equal to that of the cytochalasin B binding site in the preparation. A gel-filtration chromatography of band 4.5 photolabeled with [3H]NBTI and [3H]cytochalasin B identified three polypeptides of apparent Mr 55,000, 50,000 and 40,000. Of these, the 55 kDa polypeptide was specifically labeled by cytochalasin B (p55GT), indicating that it is a glucose transporter. Both the 50 and 40 kDa polypeptides were labeled with NBTI at low ligand concentrations (less than 0.1 microM), which was abolished by an excess (20 microM) of nitrobenzylthioguanosine, indicating that they are two forms (p50NT and p40NT, respectively) of the high affinity NBTI binding protein or nucleoside transporter. At higher (not less than 10 microM) NBTI concentrations, however, p55GT was also labeled with NBTI, indicating that the low-affinity NBTI binding is due to a glucose transporter. Treatment of band 4.5 with trypsin reduced the p50NT labeling with a concomitant and stoichiometric increase in the p40NT NBTI labeling without affecting the high-affinity NBTI binding of the preparation. These findings indicate that the nucleoside transporter is slightly smaller in mass than the glucose transporter and that trypsin digestion produces a truncated nucleoside transporter of apparent Mr 40,000 which retains the high-affinity NBTI binding activity of intact nucleoside transporter. Both p55GT and p50 NT were coeluted in a major protein fraction, P1 in the chromatography, while p40NT was eluted separately as a minor protein fraction, P1a. All three polypeptides formed mixed dimers, which were eluted in a fraction PO. We have purified and partially characterized the truncated nucleoside transporter, p40NT. The purified p40NT may be useful for biochemical characterization of the nucleoside transporter.     

Identification of the D-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to identify, the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [3H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for D-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable site. If 280 nM (40000 microunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

Evidence that forskolin binds to the glucose transporter of human erythrocytes

Binding of [4-3H]cytochalasin B and [12-3H]forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of [12-3H]forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of [12-3H]forskolin and [4-3H]cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of [4-3H]cytochalasin B or [12-3H]forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either [4-3H]cytochalasin B or [12-3H]forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.     

Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution.

The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.     

Monoclonal antibodies to the glucose transporter from human erythrocytes. Identification of the transporter as a Mr = 55,000 protein

Using the preparation of purified glucose transporter from human erythrocytes as antigen, we have prepared and characterized six monoclonal antibodies. Three of these antibodies have been shown to be to the glucose transporter by several criteria: they immunoprecipitate the transport activity, the cytochalasin B binding activity, and 75% of the protein from the solubilized purified preparation. The remaining three antibodies were shown to recognize the same polypeptide by a Western blot procedure. All of the antibodies reacted with the deglycosylated transporter and are thus against peptide determinants; most bound to the cytoplasmic domain of the transporter. The antibodies exhibited a range of effects on cytochalasin B binding, from slight enhancement to modest inhibition to strong inhibition; for this reason they must bind to at least three different epitopes. Western blot analysis of erythrocyte membranes prepared in the presence of protease inhibitors showed that all six antibodies bound to a polypeptide of average Mr = 55,000. Moreover, by immunological assay this polypeptide accounted for 5.3% of the membranes protein, a value similar to that given by cytochalasin B binding. Thus, the proposal that the native transporter is a Mr = 100,000 polypeptide is highly unlikely. The antibodies also react with the glucose transporter in other human cell types, but not with that in rodent or avian cells.     

The monosaccharide transport system of the human erythrocyte. Orientation upon reconstitution

Treatment of intact human erythrocytes with trypsin had no effect upon either the rate of hexose transport or the binding of cytochalasin B to the transport system. In contrast, proteolysis of inside-out vesicles prepared from human erythrocyte membranes inactivated both hexose transport and cytochalasin B binding. When purified hexose transporter, reconstituted into phospholipid vesicles of undetermined size, was treated with trypsin, approx. 50% of the cytochalasin B binding activity was lost. This loss correlated with a decrease in the amount of the transporter polypeptide, as assayed by gel electrophoresis. These results show that the orientation of the transporter can be established through trypsin treatment in conjunction with cytochalasin B binding. Small unilamellar vesicles containing transporter were prepared by sonication of larger species and by a cycle of cholate solubilization and removal of the detergent. In the former case, the transporter orients almost randomly, whereas in the latter approx. 75% of the transporters have the cytoplasmic domain extemal.     

The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites.

At any instant, the human erythrocyte sugar transporter presents at least one sugar export site but multiple sugar import sites. The present study asks whether the transporter also presents more than one sugar exit site. We approached this question by analysis of binding of [3H]cytochalasin B (an export conformer ligand) to the human erythrocyte sugar transporter and by analysis of cytochalasin B modulation of human red blood cell sugar uptake. Phloretin-inhibitable cytochalasin B binding to human red blood cells, to human red blood cell integral membrane proteins, and to purified human red blood cell glucose transport protein (GluT1) displays positive cooperativity at very low cytochalasin B levels. Cooperativity between sites and K(d(app)) for cytochalasin B binding are reduced in the presence of intracellular ATP. Red cell sugar uptake at subsaturating sugar levels is inhibited by high concentrations of cytochalasin B but is stimulated by lower (<20 nM) concentrations. Increasing concentrations of the e1 ligand forskolin also first stimulate then inhibit sugar uptake. Cytochalasin D (a cytochalasin B analogue that does not interact with GluT1) is without effect on sugar transport over the same concentration range. Cytochalasin B and ATP binding are synergistic. ATP (but not AMP) enhances [3H]cytochalasin B photoincorporation into GluT1 while cytochalasin B (but not cytochalasin D) enhances [gamma-32P]azidoATP photoincorporation into GluT1. We propose that the red blood cell glucose transporter is a cooperative tetramer of GluT1 proteins in which each protein presents a translocation pathway that alternates between uptake (e2) and export (e1) states but where, at any instant, two subunits must present uptake (e2) and two subunits must present exit (e1) states.     

Molecular characterization of the human erythrocyte anion transport protein in octyl glucoside

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was purified in the presence of the nonionic detergent octyl glucoside. A molecular characterization was carried out to investigate whether the native structure of the protein was retained in the presence of this detergent. Band 3 bound octyl glucoside below the critical micelle concentration (cmc) of the detergent, approaching saturation above the cmc. At 40 mM octyl glucoside, close to saturating concentrations, 0.64 g of octyl glucoside is bound per gram of band 3 protein, corresponding to 208 molecules of detergent bound per monomer of band 3. Sedimentation velocity and gel filtration studies, performed at 40 mM octyl glucoside, indicated that the band 3-octyl glucoside complex had an average molecular weight of 1.98 X 10(6), which corresponds to a dodecamer. Sedimentation equilibrium experiments confirmed that band 3 in octyl glucoside exists in a heterogeneous and high oligomeric state. This high oligomeric state did not change dramatically over octyl glucoside concentrations ranging from 6 to 60 mM. The circular dichroism spectrum of band 3 changed only slightly over this range of octyl glucoside concentrations. The alpha-helical and beta-sheet contents of band 3 in 2 mM octyl glucoside were calculated to be 40% and 27%, respectively, indicating that no gross alteration in the secondary structure of the protein had occurred in octyl glucoside. The ability of band 3 to bind 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), a potent inhibitor (Ki = 1 microM) of anion transport, was measured to assess the integrity of the inhibitor binding site of the protein in octyl glucoside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and partial purification of liver glucose transporter GLUT2

GLUT2, the major facilitative glucose transporter isoform expressed in hepatocytes, pancreatic beta-cells, and absorptive epithelial cells, is unique not only with its low affinity and broad substrate specificity as a glucose transporter, but also with its implied function as a glucose-sensor. As a first essential step toward structural and biochemical elucidation of these unique, GLUT2 functions, we describe here the differential solubilization and DEAE-column chromatography of rat hepatocyte GLUT2 protein and its reconstitution into liposomes. The reconstituted GLUT2 bound cytochalasin B in a saturable manner with an apparent dissociation constant (K(d)) of 2.3 x 10(-6) M and a total binding capacity (B(T)) of 8.1 nmol per mg protein. The binding was completely abolished by 2% mercury chloride, but not affected by cytochalasin E. Significantly, the binding was also not affected by 500 mM D-glucose or 3-O-methyl D-glucose (3OMG). The purified GLUT2 catalyzed mercury chloride-sensitive 3OMG uptake, and cytochalasin B inhibited this 3OMG uptake. The inhibition was dose-dependent with respect to cytochalasin B, but was independent of 3OMG concentrations. These findings demonstrate that our solubilized GLUT2 reconstituted in liposomes is at least 60% pure and functional, and that GLUT2 is indeed unique in that its cytochalasin B binding is not affected by its substrate (D-glucose) binding. Our partially purified GLUT2 reconstituted in vesicles will be useful in biochemical and structural elucidation of GLUT2 as a glucose transporter and as a possible glucose sensor.     

Cytochalasin B does not serve as a marker of glucose transport in rabbit erythrocytes

Glucose inhibitable cytochalasin B binding to erythrocyte membranes has been used as a marker of the glucose transporter. Glucose transport and cytochalasin B binding in rabbit erythrocytes differ from those activities found in human erythrocytes. We evaluated the uptake of 3-0-methylglucose and found similar Km (4.81 +/- 1.20 mM (SEM) and 6.59 +/- 0.72 mM) though significantly different Vmax (5.2 +/- 0.7 nM . min-1/10(9) cells and 234 +/- 13 nM X min -1/10(9) cells, p less than 0.001) for rabbit and human erythrocytes, respectively. Equilibrium binding of cytochalasin B to human erythrocyte membranes demonstrates a high affinity cytochalasin B binding site (Kd 38.6 +/- 22.7 nM) which is displaced by glucose. No comparable glucose inhibitable cytochalasin B site exists for rabbit erythrocyte membranes. Photoaffinity labeling of cytochalasin B confirms the presence of a glucose inhibitable cytochalasin B binding site in human, but not rabbit erythrocyte membranes. Cytochalasin B binding is a useful method in the identification of the glucose transporter in human cells, but the technique may be less useful in other species.     

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.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Improved preparation of the integral membrane proteins of human red cells, with special reference to the glucose transporter

Human red cell membranes were isolated and partially stripped of peripheral proteins by gel filtration of hemolysates on a Sepharose CL-4B column at pH 8 connected in tandem to a Sepharose CL-6B column at pH 10.5. The eluted material was washed by centrifugations, once at pH 10.5 and twice at pH 12. In this way, water-soluble proteins and peripheral membrane proteins were thoroughly removed, and 0.2 g of integral membrane proteins could be prepared within 10 h from 0.2 litre of red cells. The exposure to high pH did not lower the D-glucose transport activity, and electrophoretically pure glucose transport protein could be isolated from this preparation. Gel filtration in sodium dodecyl sulfate separated the integral membrane components into four fractions, one of them containing 4.5-material; gel electrophoresis showed about 14 zones and two-dimensional electrophoresis resolved up to 100 mostly minor components, among which the glucose transporter focused around pH 7. However, purified glucose transporter focused around pH 8. Glucose and nucleoside transport proteins were co-purified in active form on DEAE-cellulose and a fraction isolated by adsorption to Mono Q was used for immunization of mice and production of monoclonal antibodies. One hybridoma produced antibodies that reacted with material in the 4.5-region, possibly the glucose transport protein, and not with band 3-material. Upon two-dimensional electrophoresis of integral membrane components that had been solubilized with octyl glucoside the immunoreactive and the silver-stained 4.5-material focused in a broad range from pH 6 to pH 9. A possible explanation for this heterogeneity might be interaction between the glucose and nucleoside transport proteins and negatively charged lipids.     

Characterization and identification of the glucose transporter of human erythrocytes

The glucose transporter was purified from human erythrocytes (Kasahara, M. and Hinkle, P.C. (1977) J. Biol. Chem. 252, 7384–7390). The following results support the conclusion that a major protein in the purified transporter fraction, zone 4.5 is the glucose transporter (or a part of the transporter) and is different from band 3: (1) peptide maps of zone 4.5 were similar throughout the broad band in sodium dodecyl sulfate-gel electrophoresis and were different from those of band 3, (2) specific binding of cytochalasin B was found to the transporter fraction, but not to a band 3 fraction, (3) the N-terminal amino acid analysis of the transporter fraction showed a single N-terminal of lysine, whereas the band 3 fraction showed no clear N-terminal, and (4) the rabbit antibody raised against the transporter fraction formed a precipitation line with the transporter fraction, but not with the band 3 fraction. A filtration apparatus was devised for quick and accurate measurement of cytochalasin B binding, with which results comparable to those from equilibrium dialysis were obtained.     

D-Glucose-sensitive and -insensitive cytochalasin B binding proteins from microvillous plasma membranes of human placenta. Identification of the D-glucose transporter

Cytochalasin B was found to bind to at least two distinct sites in human placental microvillous plasma membrane vesicles, one of which is likely to be intimately associated with the glucose transporter. These sites were distinguished by the specificity of agents able to displace bound cytochalasin B. [3H]Cytochalasin B was displaceable at one site by D-glucose but not by dihydrocytochalasin B; it was displaceable from the other by dihydrocytochalasin B but not by D-glucose. Some binding which could not be displaced by D-glucose + cytochalasin B binding site. Cytochalasin B can be photoincorporated into specific binding proteins by ultraviolet irradiation. D-Glucose specifically prevented such photoaffinity labeling of a microvillous protein component(s) of Mr = 60,000 +/- 2000 as determined by urea-sodium dodecyl sulfate acrylamide gel electrophoresis. This D-glucose-sensitive cytochalasin B binding site of the placenta is likely to be either the glucose transporter or be intimately associated with it. The molecular weight of the placental glucose transporter agrees well with the most widely accepted molecular weight for the human erythrocyte glucose transporter. Dihydrocytochalasin B prevented the photoincorporation of [3H]cytochalasin B into a polypeptide(s) of Mr = 53,000 +/- 2000. This component is probably not associated with placental glucose transport. This report presents the first identification of a sodium-independent glucose transporter from a normal human tissue other than the erythrocyte. It also presents the first molecular weight identification of a human glucose-insensitive high-affinity cytochalasin B binding protein.     

Exofacial photoaffinity labelling of the human erythrocyte sugar transporter

The 4-azidosalicylate derivative of 1,3-bis(D-mannos-4'-yloxy)-2-[2-3H]propylamine (ASA-[2-3H]BMPA) has been tested as a photoaffinity label for the sugar transporter in human erythrocytes. When photolysed in the presence of intact erythrocytes, ASA-[2-3H]BMPA covalently binds to the exofacial surface of the transporter. This labelled protein appears as a broad band in the 4.5 region in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The peak of radiolabel incorporation gives an apparent Mr of approx. 50 000 on 5-20% acrylamide gels. The binding is 80% inhibitable by 320 mM 4,6-O-ethylidene-D-glucose, by 320 mM D-glucose and by 50 microM cytochalasin B. Photoirradiation of a saturating concentration of ASA-BMPA in the presence of erythrocytes results in a 25-30% loss of D-galactose transport activity. From transport inactivation data and estimations of the amount of ASA-[2-3H]BMPA binding to the transporter it is calculated that there are approx. 220 000 exofacial hexose-transport binding sites per erythrocyte. The labelling of the transporter has been carried out using freshly drawn blood and 4-weeks-old transfusion blood. No change in the binding profile on SDS-polyacrylamide gel electrophoresis was observed. Proteolytic digestion of the ASA-[2-3H]BMPA-labelled transporter with either trypsin or alpha-chymotrypsin results in the appearance of a labelled 19 kDa fragment on SDS-polyacrylamide gel electrophoresis.