Reaction of an exofacial sulfhydryl group on the erythrocyte hexose carrier with an impermeant maleimide. Relevance to the mechanism of hexose transport

The presence of a reactive exofacial sulfhydryl on the human erythrocyte hexose carrier was used to test several predictions of the alternating conformation or one-site model of transport. The cell-impermeant glutathione-maleimide-I (GS-Mal) irreversibly inhibited hexose entry by decreasing the transport Vmax. This effect was potentiated by phloretin and maltose but decreased by cytochalasin B, indicating that under the one-site model the external sulfhydryl is on the outward-facing carrier but that it does not overlap with the exofacial substrate-binding site. Incubation of erythrocytes with maltose competitively inhibited the binding of [3H]cytochalasin B to the inward-facing carrier (Ki = 40 mM). Furthermore, both equilibrium cytochalasin B binding and its photolabeling of the band 4.5 carrier protein were decreased in ghosts prepared from GS-Mal-treated cells. Thus induction of an outward-facing carrier conformation with either maltose or GS-Mal caused the endofacial substrate-binding site to disappear. Dose-response studies of GS-Mal treatment of intact cells suggested that some functional carriers lack a reactive external sulfhydryl, which can be partially regenerated by pretreatment with excess cysteine. These data provide direct support for the one-site model of transport and further define the role of the external sulfhydryl in the transport mechanism.     

Differential labeling of the erythrocyte hexose carrier by N-ethylmaleimide: correlation of transport inhibition with reactive carrier sulfhydryl groups

Inhibition of hexose transport by N-ethylmaleimide was studied with regard to alkylation of different types of sulfhydryl group on the hexose carrier of the human erythrocyte. Uptake of 3-O-methylglucose was progressively and irreversibly inhibited by N-ethylmaleimide, with a half-maximal effect at 10-13 mM. A sulfhydryl group known to exist on the exofacial carrier was not involved in transport inhibition by N-ethylmaleimide, since reversible protection of this group by the impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) had no effect on the ability of N-ethylmaleimide to inhibit transport, or on its ability to decrease the affinity of the exofacial carrier for maltose. Nevertheless, the exofacial sulfhydryl was quite reactive with N-ethylmaleimide, since it was possible using a differential labeling technique to specifically label this group in protein-depleted ghosts with a half-maximal effect at 0.3 mM N-[3H]ethylmaleimide, and to localize it to the Mr 19,000 tryptic carrier fragment. Transport inhibition by N-ethylmaleimide correlated best with labeling of a single cytochalasin B-sensitive internal sulfhydryl group on the glycosylated Mr 23,000-40,000 tryptic fragment of the carrier, which was half-maximally labeled at about 4 mM reagent. Whereas N-ethylmaleimide readily alkylates the exofacial carrier sulfhydryl, it inhibits transport by reacting with at least one internal carrier sulfhydryl located on the glycosylated tryptic carrier fragment.     

Co-operativity in seminal ribonuclease function. Kinetic studies.

Maltose-maleimide was synthesized as a potential affinity label for the facilitative hexose carrier with selectivity for exofacial sulphydryl groups. This reagent, although probably a mixture of isomers, did not significantly penetrate the plasma membrane of human erythrocytes at concentrations below 5 mM at 37 degrees C. When allowed to react to completion, it irreversibly inhibited the uptake of 3-O-methylglucose, with a half-maximal response at about 1.5-2.0 mM-reagent. The rate of transport inactivation was a saturable function of the maltose-maleimide concentration. Studies of reaction kinetics and effects of known transport inhibitors demonstrated that irreversible reaction occurred on the exofacial outward-facing carrier, although not at a site involved in substrate binding. Reaction of intact erythrocytes with [14C]maltose-maleimide resulted in labelling of a broad band 4.5 protein of Mr (average) 45,000-66,000 in electrophoretic gels. This protein was very likely the hexose carrier, since its labelling was inhibited by cytochalasin B. Exofacial band 4.5 labelling was stoichiometric with respect to transport inhibition, yielding an estimated 300,000 carriers/cell. These results suggest that the exofacial sulphydryl which reacts with maltose-maleimide is distinct from the substrate binding site on the hexose carrier, but that it confers substantial labelling selectivity to impermeant maleimides. Additionally, the high efficiency of carrier labelling obtained with maltose-maleimide is useful in quantifying numbers of carriers in whole cells.     

Reaction of the glucose carrier of erythrocytes with sodium tetrathionate: Evidence for inward-facing and outward-facing carrier conformations

Summary Sodium tetrathionate reacts with the glucose carrier of human erythrocytes at a rate which is greatly altered in the presence of competitive inhibitors of glucose transport. Inhibitors bound to the carrier on the outer surface of the membrane, either at the substrate site (maltose) or at the external inhibition site (phloretin and phlorizin), more than double the reaction rate. Inhibitors bound at the internal inhibition site (cytochalasin B and androstenedione), protect the system against tetrathionate. After treatment with tetrathionate, the maximum transport rate falls to less than one-third, and the properties of the binding sites are modified in unexpected ways. The affinity of externally bound inhibitors rises: phloretin is bound up to seven times more strongly and phlorizin and maltose twice as strongly. The affinity of cytochalasin B, bound at the internal inhibition site, falls to half while that of androstenedione is little changed. The affinity of external glucose falls slightly. Androstenedione prevents both the fall in transport activity and the increase in phloretin affinity produced by tetrathionate. An inhibitor of anion transport has no effect on the reaction. The observations support the following conclusions: (1) Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. (2) The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. (3) The thiol group with which tetrathionate is presumed to react is not present in either the substrate site or the internal or external inhibitor site. (4) In binding asymmetrically to the carrier, a reversible inhibitor shifts the carrier partition between inner and outer forms and thereby raises or lowers the rate of tetrathionate reaction with the system. (5) Reaction with tetrathionate converts the carrier to an altered state in which the conformation at all three binding sites is changed and the rate of carrier reorientation is reduced.     

Photolabeling of the human erythrocyte glucose carrier with androgenic steroids

Androgenic steroids, which are potent inhibitors of facilitated hexose transport in human erythrocytes, were tested as possible natural photolabels of the hexose carrier protein. Androstenedione, which inhibited 3-O-methylglucose uptake half-maximally at 30-50 microM (EC50), was the most potent inhibitor of the photolabile steroids tested. It appeared to interact directly with the carrier, since it (1) inhibited equilibrium [3H]cytochalasin B binding to high affinity D-glucose-sensitive sites in both intact cells (EC50 = 63 microM) and protein-depleted ghosts (EC50 = 61 microM), (2) inhibited cytochalasin B photolabeling of the band 4.5 carrier region in electrophoretic gels of protein-depleted ghosts (EC50 = 50 microM), and (3) underwent photoincorporation into the same gel region in a D-glucose- and cytochalasin B-sensitive fashion. However, Dixon plots for inhibition of both cytochalasin B binding and transport were upward-curving, indicating the binding of more than one molecule of androstenedione to the carrier. The photoincorporation of androstenedione into band 4.5 protein was both time- and concentration-dependent, and not associated with damage to unlabeled carrier. It probably occurred by activation of the alpha, beta-unsaturated ketone on the steroid rather than indirectly by photoactivation of a group on the carrier protein, as occurs with cytochalasin B. Although androstenedione may bind to more than one region of the carrier, as well as to other non-carrier proteins, tryptic digestion of photolabeled ghosts produced a labeled Mr = 18,000-20,000 fragment, the labeling of which was inhibited by cytochalasin B, and which had an electrophoretic mobility similar to the major labeled tryptic fragment in cytochalasin B-labeled ghosts. These data suggest that androstenedione interacts directly with the hexose carrier and that it or other similar naturally photolabile steroids may serve as useful probes for structural dissection of the carrier protein.     

Inhibition of hexose transport in the human erythrocyte by 5, 5′-dithiobis(2-nitrobenzoic acid): Role of an exofacial carrier sulfhydryl group

Summary The sulfhydryl reagent 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1mm DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10mm cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120mm accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 m cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfydryl, but reaction of this group may not always lock the carrier in an outward-facing conformation.     

Interaction of a permeant maleimide derivative of cysteine with the erythrocyte glucose carrier. Differential labelling of an exofacial carrier thiol group and its role in the transport mechanism.

S-(Bismaleimidomethyl ether)cysteine (Cys-Mal) was synthesized as a probe for reactive thiol groups on the erythrocyte glucose carrier. Although Cys-Mal entered cells, its reaction with intracellular GSH prevented alkylation of endofacial membrane proteins, limiting its effect to the cell surface at concentrations below 5 mM. Cys-Mal irreversibly inhibited hexose transport half-maximally at 1.5 mM by decreasing the maximal rate of transport, with no effect on the affinity of substrate for the carrier. Reaction occurred with the outward-facing form of the carrier, but did not affect the ability of the carrier to change orientation. In intact cells, several exofacial proteins were labelled by [35S]Cys-Mal, including the band-4.5 glucose carrier, the labelling of which occurred on a single site sensitive to transport inhibitors. The reactive exofacial group was a thiol group, since both transport inhibition and band-4.5 labelling by Cys-Mal were abolished by the thiol-specific and impermeant compound 5,5'-dithiobis(2-nitrobenzoic acid). Selectivity for carrier labelling in cells was increased by a double differential procedure, which in turn allowed localization of the exofacial thiol group to the Mr 18,000-20,000 membrane-bound tryptic carrier fragment. In protein-depleted ghosts the exofacial thiol group was preferentially labelled at low concentrations of [35S]Cys-Mal, whereas with the reagent at 10 mM the Mr 26,000-45,000 tryptic carrier fragment was also labelled. Cys-Mal should be useful in the study of carrier thiol-group location and function.     

Cytochalasin B-binding proteins in rabbit erythrocyte membranes and their post-natal change in relation to the glucose carrier activity

Two distinct, carrier-mediated glucose uptake processes, a fast, cytochalasin B-sensitive and a slow, cytochalasin B-insensitive flux are identified in parallel in newborn rabbit erythrocytes. The fast, cytochalasin B-sensitive carrier function disappears as rabbits age, and only the slow cytochalasin B-insensitive carrier function is observed with adult rabbit erythrocytes.Three different cytochalasin B binding sites are distinguished in newborn rabbit erythrocytes; a glucose-sensitive site (site I), a cytochalasin E-sensitive site (site II), and a site insensitive to both glucose and cytochalasin E. With adult rabbit erythrocytes, only a cytochalasin E-sensitive site is detected. The glucose-sensitive site disappears as rabbits age, with a time course which is comparable to that of the disappearance of the cytochalasin B-sensitive glucose carrier function. The cytochalasin E-sensitive cytochalasin B binding site does not increase during this change, thus the disappearance of the glucose-sensitive site is not due to its conversion to a cytochalasin E-sensitive site. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of rabbit erythrocyte ghosts revealed a partial decrease in each of the membrane polypeptides of approximate molecular weights of 240 000, 160 000 and 50 000 as rabbits aged. It is concluded that the cytochalasin B-sensitive glucose carrier of fetal rabbit erythrocytes, like that of the human erythrocyte, is tightly associated with the site I cytochalasin B-binding protein, while the cytochalasin B-insensitive glucose carrier, operative in adult rabbit erythrocytes, is not.     

Affinity labeling of forskolin-binding proteins. Comparison between glucose carrier and adenylate cyclase

An [125I]iodoazidosalicylic acid derivative of forskolin was synthesized for identification of the diterpene's binding sites on the catalytic subunit of adenylate cyclase and on glucose transport proteins. The affinity label was selectively incorporated into proteins of Mr 40,000-60,000 in membranes from human erythrocytes and from various other tissues. The iodoazidosalicylic acid derivative also specifically labeled the catalytic moiety of adenylate cyclase from rabbit myocardial membranes. However, the structural requirements of the two forskolin-binding sites must be different, since the affinity of the photolabel for the glucose carriers is much higher than that for the cyclase catalyst. Furthermore, the label is readily competed with by D-glucose and cytochalasin B for its binding site on the glucose carrier but not on adenylate cyclase.     

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.     

Inhibition of hexose transport by adenosine derivatives in human erythrocytes

The human erythrocyte membrane carriers for hexoses and nucleosides have several structural features in common. In order to assess functional similarities, the effects of adenosine derivatives on hexose transport and cytochalasin B binding sites were studied. Adenosine inhibited zero-trans uptake of 3-O-methylglucose half-maximally at 5 mM, while more hydrophobic adenosine deaminase-resistant derivatives were ten- to 20-fold more potent transport inhibitors. However, degradation of adenosine accounted for very little of this difference in potency. Hexose transport was rapidly inhibited by N6-(L-2-phenylisopropyl)adenosine at 5 degrees C in a dose-dependent fashion (EC50 = 240 microM), to lower the transport Vmax without affecting the Km. A direct interaction with the carrier protein was further indicated by the finding that N6-(L-2-phenylisopropyl)adenosine competitively inhibited [3H]cytochalasin B binding to erythrocytes (Ki = 143 microM) and decreased [3H]cytochalasin B photolabeling of hexose carriers in erythrocyte ghosts. The cross-reactivity of adenosine and several of its derivatives with the hexose carrier suggests further homologies between the carriers for hexoses and nucleosides, possibly related to their ability to transport hydrophilic molecules through the lipid core of the plasma membrane.     

Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.     

The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes

The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces, is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.     

The human erythrocyte sugar transporter presents two sugar import sites

The human erythrocyte sugar transporter presents sugar import (e2) and sugar export (e1) sites simultaneously. This study asks whether the sugar transporter exposes only one or multiple import sites. We approached this question by analysis of cytochalasin B binding to the human erythrocyte sugar export site in the presence of sugars that bind to the sugar import site. Extracellular maltose does not enter human erythrocytes. High concentrations of maltose (1-100 mM) inhibit cytochalasin B binding to human red cells. Low concentrations (25-500 microM) increase the level of erythrocyte cytochalasin B binding. Maltose modulation of cytochalasin B binding is mediated by altered affinity of sugar export sites for cytochalasin B. Similar results are obtained with other cell-impermeant inhibitors of sugar uptake. Extracellular D-glucose (a transported sugar) stimulates cytochalasin B binding at low D-glucose concentrations (10-250 microM), but this effect is lost at higher concentrations. Intracellular D-glucose inhibits cytochalasin B binding. Low concentrations of extracellular maltose and other nontransported inhibitors stimulate 3-O-methylglucose uptake in erythrocytes. Higher sugar concentrations (1-100 mM) inhibit transport. These data support the hypothesis that the erythrocyte sugar transporter presents two sugar import sites and at least one sugar export site. This conclusion is consistent with the proposed oligomeric structure of the sugar transporter, a complex of four GluT1 proteins in which each subunit presents a translocation pathway.     

Cytochalasin B and the kinetics of inhibition of biological transport: a case of asymmetric binding to the glucose carrier

Cytochalasin B inhibits glucose transport in human erythrocytes by competing with glucose for the carrier on the inner surface of the cell membrane, but there is no cytochalasin site associated with the outware-facing form of the carrier. Such asymmetry may be demonstrated by zero trans exit and entry experiments, whereas Sen-Widdas exit experiments are not easily interpretable. The orientation of the transport system appears to be reversed in certain other cell types: chich embryo fibroblasts, Novikoff hepatoma cells and HeLa cells. Here the cytochalasin site is present in the external but not internal carrier form.     

Cytochalasin B and the kinetics of inhibition of biological transport. A case of asymmetric binding to the glucose carrier

Cytochalasin B inhibits glucose transport in human erythrocytes by competing with glucose for the carrier on the inner surface of the cell membrane, but there is no cytochalasin site associated with the outward-facing form of the carrier. Such asymmetry may be demonstrated by zero trans exit and entry experiments, whereas Sen-Widdas exit experiments are not easily interpretable. The orientation of the transport system appears to be reversed in certain other cell types: chick embryo fibroblasts, Novikoff hepatoma cells and HeLa cells. Here the cytochalasin site is present in the external but not internal carrier form.     

Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site

Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport [ Cloherty, E. K., Levine, K. B., and Carruthers, A. ((2001)) The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry 40 ((51)) 15549-15561]. This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, endofacial binding sites for CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogues, asks what structural features of endofacial site ligands determine binding site affinity and cooperativity. Like CB, FSK competitively inhibits exchange 3-O-methylglucose transport (sugar uptake in cells containing intracellular sugar) but noncompetitively inhibits sugar uptake into cells lacking sugar at 4 °C. This refutes the hypothesis that FSK binds at GLUT1 endofacial and exofacial sugar binding sites. Some forskolin derivatives and cytochalasins inhibit equilibrium [(3)H]-CB binding to red cell membranes depleted of peripheral proteins at 4 °C. Others produce a moderate stimulation of [(3)H]-CB binding when introduced at low concentrations but inhibit binding as their concentration is increased. Yet other analogues modestly stimulate [(3)H]-CB binding at all inhibitor concentrations applied. These findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of models for GLUT1-mediated sugar transport and GLUT1 quaternary structure, and we evaluate the major determinants of ligand binding affinity and cooperativity.     

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

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

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.     

alpha- and beta-monosaccharide transport in human erythrocytes

Equilibrative sugar uptake in human erythrocytes is characterized by a rapid phase, which equilibrates 66% of the cell water, and by a slow phase, which equilibrates 33% of the cell water. This behavior has been attributed to the preferential transport of beta-sugars by erythrocytes (Leitch JM, Carruthers A. Am J Physiol Cell Physiol 292: C974-C986, 2007). The present study tests this hypothesis. The anomer theory requires that the relative compartment sizes of rapid and slow transport phases are determined by the proportions of beta- and alpha-sugar in aqueous solution. This is observed with D-glucose and 3-O-methylglucose but not with 2-deoxy-D-glucose and D-mannose. The anomer hypothesis predicts that the slow transport phase, which represents alpha-sugar transport, is eliminated when anomerization is accelerated to generate the more rapidly transported beta-sugar. Exogenous, intracellular mutarotase accelerates anomerization but has no effect on transport. The anomer hypothesis requires that transport inhibitors inhibit rapid and slow transport phases equally. This is observed with the endofacial site inhibitor cytochalasin B but not with the exofacial site inhibitors maltose or phloretin, which inhibit only the rapid phase. Direct measurement of alpha- and beta-sugar uptake demonstrates that erythrocytes transport alpha- and beta-sugars with equal avidity. These findings refute the hypothesis that erythrocytes preferentially transport beta-sugars. We demonstrate that biphasic 3-O-methylglucose equilibrium exchange kinetics refute the simple carrier hypothesis for protein-mediated sugar transport but are compatible with a fixed-site transport mechanism regulated by intracellular ATP and cell shape.