Kinetic properties of the reconstituted glucose transporter from human erythrocytes

The kinetic parameters of D-glucose transport in liposomes reconstituted with the purified glucose transporter were determined. Net uptake and efflux both had Km values of 0.7 to 1.2 mM and Vmax values of 1.6 mumol/mg of protein/min. Equilibrium exchange had a Km of 35 mM and a Vmax of 50 mumol/mg of protein/min. By separating the liposomes from unreconstituted protein using density centrifugation, the Vmax of exchange was increased to 86 mumol/mg of protein/min, about 3 times that of the erythrocyte membrane. Trypsin, which inhibits erythrocyte glucose transport only from the cytoplasmic side, inhibited reconstituted transport activity about 40% when added externally. With internal treatment as well, the inhibition was about 80%. This suggests that the reconstituted transporter is oriented about equally in both directions. Antibody prepared against the purified transporter inhibits transport to a maximum of about 50%, also indicating a scrambled orientation. External trypsin treatment decreased the Km for uptake and increased the Km for efflux, consistent with asymmetric kinetic parameters for the two faces of the transporter. However, the calculated Km values are lower than those reported for erythrocytes. Phloretin and diethylstilbestrol inhibit the reconstituted transporter. However, they bind to liposomes, producing anomalous results under some experimental conditions. When this binding is taken into account, phloretin inhibits completely and symmetrically. The binding accounts for the apparent asymmetric effects of phloretin reported by others. The inhibitory effects of mercuric ions are consistent with action at two classes of binding sites. Treatment with trypsin increases the sensitivity to Hg2+, indicating that the more sensitive site is on the external face of the transporter.     

Cytochalasin B interferes with conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding.

To gain insight into the mechanism of facilitated sugar transport and possible mechanisms by which glucose transporter intrinsic activity might be altered, we have investigated conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding and by the transporter inhibitor, cytochalasin B. Changes in the ability of thermolysin to digest glucose transporters present in erythrocyte ghosts were used to monitor conformational changes of the glucose transporter. The degree of protease digestion was determined by the amount of undigested glucose transporter remaining after the protease treatment, as assessed in Western blots using the glucose transporter specific monoclonal antibody 7F7.5. D-Glucose, the physiological substrate of the transporter, increased the transporter's susceptibility to cleavage by thermolysin. Nontransportable glucose analogues which bind specifically to either an internal or external glucose transporter sugar binding site also altered susceptibility of the transporter to thermolysin. Both methyl and propyl glucoside, which preferentially bind the internal sugar site, increased thermolysin susceptibility of the glucose transporter in a manner similar to that of D-glucose. In contrast, 4,6-O-ethylideneglucose, which preferentially binds the external sugar site, protected the transporter from thermolysin digestion. These results suggest that sugar binding to internal and external sugar sites induces distinct conformational changes and that the observed D-glucose effect on the susceptibility of the glucose transporter to thermolysin is due to D-glucose at equilibrium predominantly forming a complex with the internal sugar site. The protection from cleavage by thermolysin caused by external sugar binding is attenuated by the addition of an internally binding sugar.(ABSTRACT TRUNCATED AT 250 WORDS)     

An ATP-modulated specific association of glyceraldehyde-3-phosphate dehydrogenase with human erythrocyte glucose transporter

Glyceraldehyde-3-phosphate dehydrogenase was found to bind in vitro to purified, human erythrocyte glucose transporter reconstituted into vesicles. Mild tryptic digestion of the glucose transporter totally inactivated the binding, suggesting that the cytoplasmic domain of the transporter is involved in the binding to glyceraldehyde-3-phosphate dehydrogenase. The binding was abolished in the presence of antisera raised against the purified glucose transporter, further supporting specificity of this interaction. The binding was reversible with a dissociation constant (Kd) of 3.3 x 10(-6) M and a total capacity (Bt) of approximately 30 nmol/mg of protein indicating a stoichiometry of one enzyme-tetramer per accessible transporter. The binding was sensitive to changes in pH showing an optimum at around pH 7.0. KCl and NaCl inhibited the binding in a simple dose-dependent manner with Ki of 40 and 20 mM, respectively. The binding was also inhibited by NAD+ with an estimated Ki of 3 mM. ATP, on the other hand, enhanced the binding by up to 3-fold in a dose-dependent manner with an apparent Ka of approximately 6 mM. The binding was not affected by D-glucose or cytochalasin B. The binding did not affect either the glucose or cytochalasin B in binding affinities or the transport activity of the transporter. However, the enzyme was inactivated totally upon binding to the transporter. Based on these findings, we suggest that a significant portion of glyceraldehyde-3-phosphate dehydrogenase in human erythrocytes exists as an inactive form via an ATP-dependent, reversible association with glucose transporter, and that this association may exert regulatory intervention on nucleotide metabolism in vitro.     

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.     

Long term regulation of glucose transporters by insulin in mature 3T3-F442A adipose cells. Differential effects on two glucose transporter subtypes

The question of a long term regulatory role of insulin on adipocyte glucose transporter content was addressed using the differentiating or fully mature 3T3-F442A adipocytes. Glucose transport was measured in intact cells. Glucose transporter content in plasma membranes and low density microsomes (LDM) was assessed by cytochalasin B binding and Western analysis. In insulin- versus spontaneously differentiated adipocytes, glucose transport and glucose transporters content of plasma membranes and LDM were increased 5-, 4-, and 2-fold, respectively. Insulin deprivation for 24 h induced a redistribution of glucose transporters in those cells which then displayed 2-fold higher glucose transport and glucose transporter content in plasma membranes than spontaneously differentiated cells and 3-fold more glucose transporters in LDM. When fully insulin-differentiated adipocytes were insulin-deprived for 4 days, there was a marked decrease in glucose transporters in both membrane fractions that was fully reversible by reexposing the cells to insulin for 4 days. Glucose uptake changes were closely proportionate to changes in glucose transporter content of plasma membranes as assessed by an antiserum to the C-terminal peptide of the erythrocyte/HepG2/brain-type glucose transporter. When Western blots were immunoblotted with 1F8 monoclonal antibody, specific for glucose transporter in insulin responsive tissues, an abundant immunoreactive protein was detected in both plasma membranes and LDM but the amount of this glucose transporter did not change with insulin exposure in any membrane fractions. In conclusion, insulin plays a long term regulatory role on cultured adipocyte glucose transporter content through a selective effect on the erythrocyte/HepG2/brain-type glucose transporter.     

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.     

Critical parameters for functional reconstitution of glucose transport in Trypanosoma brucei membrane vesicles

The glucose transporter of Trypanosoma brucei was reconstituted by incorporating Escherichia coli phospholipid liposomes into detergent-solubilised trypanosome membranes. Proteoliposome vesicles were formed by detergent dilution and used in glucose-uptake assays. The minima for functional reconstitution of the glucose transporter were established and used to probe the mechanism of glucose transport. The uptake pattern of radiolabelled glucose showed a counterflow transient at about 3 s, after which the sugar equilibrated across the proteoliposomal membrane. This observation is consistent with a facilitated transporter. There was a six-fold increase in the initial rate of glucose uptake compared to non-reconstituted or native membranes. In addition, the transporter exhibited stereospecificity to D-glucose but poorly transported L-glucose. Directionality, stereoselectivity or substrate specificity and cis-inhibition by phloridzin were therefore the main criteria for validation of glucose transport. The observed counterflow transient also provided further evidence for a facilitated glucose transporter within the trypanosome plasma membrane, and was the single most important criterion for this assertion. A stoichiometry of 0.78 mol of glucose per mol of transporter was estimated.     

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.     

A D-Glucose-Sensitive Cytochalasin B Binding Component of Cerebral Microvessels

The technique of photoaffinity labelling with [4-3H]cytochalasin B was applied to osmotically lysed cerebral microvessels isolated from sheep brain. Cytochalasin B was photo-incorporated into a membrane protein of average apparent Mr 53,000. Incorporation of cytochalasin B was inhibited by D-glucose, but not by L-glucose, which strongly suggests that the labelled protein is, or is a component of, the glucose transporter of the blood-brain barrier. Investigation of noncovalent [4-3H]cytochalasin B binding to cerebral microvessels by equilibrium dialysis indicated the presence of a single set of high-affinity binding sites with an association constant of 9.8 +/- 1.7 (SE) microM-1. This noncovalent binding was inhibited by D-glucose, with a Ki of 23 mM. These results provide preliminary identification of the glucose transporter of the ovine blood-brain barrier, and reveal both structural and functional similarities to the glucose transport protein of the human erythrocyte.     

Reconstitution of the glucose transporter from bovine heart

Reconstitution of the glucose transporter from heart should be useful as an assay in its purification and in the study of its regulation. We have prepared plasma membranes from bovine heart which display D-glucose reversible binding of cytochalasin B (33 pmol sites/mg protein; Kd = 0.2 muM). The membrane proteins were reconstituted into liposomes by the freeze-thaw procedure. Reconstituted liposomes showed D-glucose transport activity which was stereospecific, saturable and inhibited by cytochalasin B, phloretin, and mercuric chloride. Compared to membrane proteins reconstituted directly, proteins obtained by dispersal of the membranes with low concentrations of cholate or by cholate solubilization showed 1.2- or 2.3-fold higher specific activities for reconstituted transport, respectively. SDS-polyacrylamide gel electrophoresis followed by electrophoretic protein transfer and labeling with antisera prepared against the human erythrocyte transporter identified a single band of about 45 kDa in membranes from both dog and bovine hearts, a size similar to that reported for a number of other glucose transporters in various animals and tissues.     

Inhibition of glucose transport in human erythrocytes by ubiquinone Q0

Searches of the protein data bases revealed limited homologies between several regions of the human erythrocyte glucose transporter containing a relative abundance of hydrogen-bonding amino-acid side chains, and proteins of the NADH-ubiquinone oxidoreductase family. This raised the possibility the binding sites for glucose and ubiquinone may be similar in the respective proteins. Experimental studies demonstrated that ubiquinone Q0 does in fact inhibit both glucose entry and glucose exit in human erythrocytes with kinetics consistent with the existence of ubiquinone binding sites at both the exofacial and endofacial sides of the transporter. Glucose transport was also inhibited by the water-soluble tryptophan-inactivating agent, dimethyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide, and this is consistent with the presence of tryptophan residues in two of the exofacial amino-acid sequences proposed as candidates for involvement in glucose binding sites.     

Localization of the forskolin photolabelling site within the monosaccharide transporter of human erythrocytes.

Chemical and proteolytic digestion of intact erythrocyte glucose transporter as well as purified transporter protein has been used to localize the derivatization site for the photoaffinity agent 3-[125I]iodo-4-azido-phenethylamino-7-O-succinyldeacetylforskol in [( 125I]IAPS-forskolin). Comparison of the partial amino acid sequence of the labelled 18 kDa tryptic fragment with the known amino acid sequence for the HepG2 glucose transporter confirmed that the binding site for IAPS-forskolin is between the amino acid residues Glu254 and Tyr456. Digestion of intact glucose transporter with Pronase suggests that this site is within the membrane bilayer. Digestion of labelled transporter with CNBr generated a major radiolabelled fragment of Mr approximately 5800 putatively identified as residues 365-420. Isoelectric focusing of Staphylococcus aureus V8 proteinase-treated purified labelled tryptic fragment identified two peptides which likely correspond to amino acid residues 360-380 and 381-393. The common region for these radiolabelled peptides is the tenth putative transmembrane helix of the erythrocyte glucose transporter, comprising amino acid residues 369-389. Additional support for this conclusion comes from studies in which [125I]APS-forskolin was photoincorporated into the L-arabinose/H(+)-transport protein of Escherichia coli. Labelling of this transport protein was protected by both cytochalasin B and D-glucose. The region of the erythrocyte glucose transporter thought to be derivatized with IAPS-forskolin contains a tryptophan residue (Trp388) that is conserved in the sequence of the E. coli arabinose-transport protein.     

Dissection of discrete kinetic events in the binding of antibiotics and substrates to the galactose-H+ symport protein,GalP, ofEscherichia coli

GalP is the membrane protein responsible for H⁺-driven uptake of D-galactose intoEscherichia coli. It is suggested to be the bacterial equivalent of the mammalian glucose transporter, GLUT1, since these proteins share sequence homology, recognise and transport similar substrates and are both inhibited by cytochalasin B and forskolin. The successful over-production of GalP to 35–55% of the total inner membrane protein ofE. coli has allowed direct physical measurements on isolated membrane preparations. The binding of the antibiotics cytochalasin B and forskolin could be monitored from changes in the inherent fluorescence of GalP, enabling derivation of a kinetic mechanism describing the interaction between the ligands and GalP. The binding of sugars to GalP produces little or no change in the inherent fluorescence of the transporter. However, the binding of transported sugars to GalP produces a large increase in the fluorescence of 8-anilino-1-naphthalene sulphonate (ANS) excited via tryptophan residues. This has allowed a binding step, in addition to two putative translocation steps, to be measured. From all these studies a basic kinetic mechanism for the transport cycle under non-energised conditions has been derived. The ease of genetical manipulation of thegalP gene inE. coli has been exploited to mutate individual amino acid residues that are predicted to play a critical role in transport activity and/or the recognition of substrates and antibiotics. Investigation of these mutant proteins using the fluorescence measurements should elucidate the role of individual residues in the transport cycle as well as refine the current model.Abbreviations GalP galactose-H⁺ transporter - AraE arabinose-H⁺ transporter - GLUT1 human erythrocyte glucose transporter requests for offprints: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2UH, UK     

Insulin responsiveness in skeletal muscle is determined by glucose transporter (Glut4) protein level.

Glucose transport in skeletal muscle is mediated by two distinct transporter isoforms, designated muscle/adipose glucose transporter (Glut4) and erythrocyte/HepG2/brain glucose transporter (Glut1), which differ in both abundance and membrane distribution. The present study was designed to investigate whether differences in insulin responsiveness of red and white muscle might be due to differential expression of the glucose transporter isoforms. Glucose transport, as well as Glut1 and Glut4 protein and mRNA levels, were determined in red and white portions of the quadriceps and gastrocnemius muscles of male Sprague-Dawley rats (body wt. approx. 250 g). Maximal glucose transport (in response to 100 nM-insulin) in the perfused hindlimb was 3.6 times greater in red than in white muscle. Red muscle contained approx. 5 times more total Glut4 protein and 2 times more Glut4 mRNA than white muscle, but there were no differences in the Glut1 protein or mRNA levels between the fibre types. Our data indicate that differences in responsiveness of glucose transport in specific skeletal muscle fibre types may be dependent upon the amount of Glut4 protein. Because this protein plays such an integral part in glucose transport in skeletal muscle, any impairment in its expression may play a role in insulin resistance.     

Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells.

Glucose transporter isoform expression was studied in the skeletal muscle-like cell line, C2C12. Northern and Western blot analysis showed that the insulin-responsive muscle/fat glucose transporter isoform, GLUT 4, was expressed in these cells at very low levels, whereas the erythrocyte isoform, GLUT 1, was expressed at readily detectable levels. Insulin did not stimulate glucose transport in this cultured muscle cell line. The C2C12 cells were then transfected separately with either GLUT 1 or GLUT 4, and stable cell lines expressing high levels of mRNA and protein were isolated. GLUT 1-transfected cells exhibited a 3-fold increase in the amount of the GLUT 1 transporter protein which was accompanied by a 2- to 3-fold increase in the glucose uptake rate. However, despite at least a 10-fold increase in GLUT 4 mRNA and protein detected after GLUT 4 cDNA transfection, the glucose uptake of these cells was unchanged and remained insulin-insensitive. By laser confocal immunofluorescence imaging, it was established that the transfected GLUT 4 protein was localized almost entirely in cytoplasmic compartments. In contrast, the GLUT 1 isoform was detected both at the plasma membrane as well as in intracellular compartments. These results suggest that acute insulin stimulation of glucose transport is not solely dependent on the presence of the insulin receptor and the GLUT 4 protein, and that the presence of some additional protein(s) must be required.     

Monoclonal antibodies to cytoplasmic domains of the acetylcholine receptor

Fourteen clonal hybridoma lines that secrete monoclonal antibodies (mabs) to the Torpedo acetylcholine receptor (AChR) have been isolated. When analyzed by an immunoreplica technique, two mabs recognized the alpha subunit, three reacted with the beta subunit, one reacted with the gamma chain, and five recognized the delta subunit. One mab failed to react with any of the subunits using this assay and two mabs recognized determinants found on both the gamma and the delta subunits. These were classified according to their reactivities with the membrane-bound Torpedo AChR. One category is comprised of mabs (including both anti-alpha mabs) that recognize extracellular epitopes. A second classification included mabs that are unable to bind the membrane-associated AChR. The third category is comprised of mabs directed against cytoplasmic epitopes of the AChR. The latter mabs, all of which recognize the gamma or delta subunits or both, bind only slightly to sealed, outside-out Torpedo vesicles. The binding is increased 10-20-fold by either alkaline extraction or treatment of the vesicles with 10 mM lithium diiodosalicylate but not by permeabilization of the vesicles with saponin. Three of the six mabs in this category react with frog muscle endplates but only if the cytoplasmic surface of the membrane is accessible.     

The human erythrocyte sugar transporter is also a nucleotide binding protein

We have previously shown that ATP interacts with an intracellular, stereoselective, regulatory site(s) on the human erythrocyte sugar transport system to modify transport function in a hydrolysis-independent manner. This present study examines the nucleotide binding properties of the human erythrocyte sugar transport system. We demonstrate by transport studies in ghosts, by nucleotide binding studies with purified transport protein by measurements of nucleotide inhibition of 8-azidoadenosine 5'-[gamma-32P]triphosphate (azido-ATP) photoincorporation into purified carrier, and by analysis of nucleotide inhibition of carboxyl-terminal peptide antisera binding to purified glucose carrier than the glucose transport protein binds (with increasing order of affinity) AMP, ADP, ATP, 5'-adenylyl imidodiphosphate (AMP-PNP), and 1,N6-ethenoadenosine 5'-triphosphate (EATP) at a single site. The carrier lacks detectable ATPase activity and GTP binding capacity. While AMP and ADP bind to the carrier protein and act as competitive inhibitors of ATP binding, these nucleotides are unable to mimic the ability of ATP, AMP-PNP, and EATP to modify the catalytic properties of the sugar transport system. Limited tryptic digestion of azido-ATP-photolabeled carrier suggests that the region of the glucose transport protein containing the intracellular cytochalasin B binding and extracellular bis(mannose) binding domains [residues 270-456; Holman, G. D., & Rees, W. D. (1987) Biochim. Biophys. Acta 897, 395-405] may also contain the intracellular ATP binding site.     

Phenylarsine oxide stimulates hexose transport in 3T3-L1 adipocytes by a mechanism other than an increase in surface transporters

Phenylarsine oxide (PAO) has been shown to exert a biphasic effect on glucose transport in 3T3-L1 adipocytes. At 10 microM, PAO activates transport threefold, but at higher concentrations an inhibition of transport is observed. In this paper we report a procedure for the subcellular fractionation of these cells which we use to examine the distribution of glucose transporters following PAO challenge. Quantitative immunoblotting showed that the glucose transporter content of the plasma membrane fraction increased with increasing PAO concentrations; a parallel increase in another insulin-responsive protein, the transferrin receptor, also occurred. However, cell-surface labeling procedures for the glucose transporter and transferrin receptor showed that PAO actually decreased the cell-surface concentrations of these proteins; the basis of this discrepancy may be that in the presence of PAO, intracellular vesicles containing these proteins associate with the plasma membrane, but do not fuse with it. The possibility that PAO modulated transport by direct interaction with the glucose transporter was investigated by examining the effects of PAO on transport in both erythrocytes and a reconstituted system of purified erythrocyte transporter in lipid vesicles. PAO was without effect on the rate of transport in these systems. The hypothesis that the stimulatory effect of PAO on transport might be due to the activation of the insulin receptor kinase activity was examined by assessing the phosphotyrosine content of the receptor and other proteins using anti-phosphotyrosine antibodies. PAO alone caused no detectable increase in receptor phosphotyrosine content. However, the combination of PAO and insulin led to the tyrosine phosphorylation of two proteins of Mr 68,000 and 57,000 which were not detected in cells treated with either PAO or insulin, and an increased phosphotyrosine content of proteins of Mr 95,000 and 165,000 when compared to cells treated with insulin alone.     

Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein

Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.     

Peptide-specific antibodies as probes of the orientation of the glucose transporter in the human erythrocyte membrane

Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots.