Selective labeling of the erythrocyte hexose carrier with a maleimide derivative of glucosamine: relationship of an exofacial sulfhydryl to carrier conformation and structure

Sulfhydryl-reactive derivatives of glucosamine were synthesized as potentially transportable affinity labels of the human erythrocyte hexose carrier. N-Maleoylglycyl derivatives of either 6- or 2-amino-2-deoxy-D-glucopyranose were the most potent inhibitors of 3-O-methylglucose uptake, with concentrations of half-maximal irreversible inhibition of about 1 mM. Surprisingly, these derivatives were very poorly transported into erythrocytes. They reacted rather with an exofacial sulfhydryl on the carrier following a reversible binding step, the latter possibly to the exofacial substrate binding site. However, their reactivity was determined primarily by access to the exofacial sulfhydryl, which, as predicted by the one-site model of transport, required a carrier conformation with the exofacial substrate binding site exposed. Once reacted, the carrier was "locked" in a conformation unable to reorient inwardly and bind cytochalasin B. In intact erythrocytes the N-maleoylglycyl derivative of 2-[3H]glucosamine labeled predominantly an Mr 45,000-66,000 protein on gel electrophoresis in a quantitative and cytochalasin B inhibitable fashion. By use of changes in carrier conformation induced by competitive transport inhibitors in a "double" differential labeling method, virtually complete selectivity of labeling of the carrier protein was achieved, the latter permitting localization of the reactive exofacial sulfhydryl to an Mr 18,000-20,000 tryptic fragment of the carrier.     

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.     

Evidence for allosteric inhibition sites in the glucose carrier of erythrocytes

2,4-Fluorodinitrobenzene and 2,3-butanedione, which irreversibly inactivate the glucose transfer system of erythrocytes, have been used as probes to determine whether the substrate site and inner and outer sites for reversible inhibitors are located in the same or different regions of the carrier. Inhibitors bound at an inhibition site exposed in the inward-facing but not the outwardfacing form of the carrier (cytochalasin B, androstendione and androstandione) protect the transport system against inactivation by 2,4-fluorodinitrobenzene. Inhibitors bound at an external inhibition site (phloretin) and substrates bound at the transfer site do not protect. In contrast inactivation by 2,3-butanedione is slightly accelerated by internally bound inhibitors, while substrates and substrate analogs bound at the transfer site protect the system. It is shown that fluorodinitrobenzene reacts in the inner inhibition site and butanedione in the substrate site; and further that these sites may be separate binding areas in the carrier linked by allosteric interaction. The consequence of this linkage is that binding of a ligand at the substrate site precludes binding of another ligand at the internal or external inhibition site.     

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.     

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.     

Characterization of the glucose transporter from human erythrocytes

The D-glucose transporter from human erythrocytes has been purified and reconstituted by Kasahara and Hinkle (J Biol Chem 252:7394–7390). Using a similar purification scheme, we have isolated the protein with 65% of the extracted phospholipid at a lipid-protein ratio of 14:1 by weight. The K_D (0.14 μM) and extent (11 nmoles/mg protein) for binding of ³H-cytochalasin B was determined by equilibrium dialysis. Glucose was a linear competitive inhibitor of binding of cytochalasin B, with an inhibition constant of 30 mM. To further characterize the protein, samples were filtered in the presence of sodium dodecyl sulfate (SDS) through Sepharose 6B to remove 95% of the lipid followed by filtration of Sephadex G150 to remove the remaining lipid and a contaminating amount of a minor, lower-molecular-weight protein. This preparation contains only 24% acidic and basic amino acids. The protein also contains 5% neutral sugars (of which 3% is galactose), 7% glucosamine, and 5% sialic acid.     

Interaction of carboplatin with carrier human erythrocytes.

The antineoplastic drug Carboplatin (CBDCA) was encapsulated in human erythrocytes by means of transient hypotonic hemolysis, followed by isotonic resealing. Up to 5 mg/ml of packed cells could be entrapped, with about 70% cell recovery. In vitro incubation of the CBDCA-loaded erythrocytes in autologous plasma caused a very slow release of the drug from the cells (12% approximately in 3 h). The encapsulation conditions, performed at a low hematocrit, in order to obtain high amounts of the drug inside the carriers, impaired the metabolic properties of the loaded erythrocytes significantly. In particular, an almost complete disappearance of GSH was observed. Analysis of the intraerythrocytic metabolism of CBDCA showed that, in spite of its relatively high stability in aqueous solutions, in hemolysates and in the loaded erythrocytes a significant percentage of CBDCA is rapidly converted to other species that still retain an antiproliferative activity in vitro. This fast conversion could be extensively inhibited by previous conversion of oxyhemoglobin to methemoglobin or carbomonoxyhemoglobin, suggesting an important role of heme iron in this process. Encapsulation of CBDCA in selectively targeted human erythrocytes may represent a therapeutic strategy for increasing the drug concentration in specific organs, notably liver.     

Asymmetric transport of a fluorescent glucose analogue by human erythrocytes

A fluorescent glucose analogue, 6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose (NBDG), was synthesized and its interactions with the hexose transport system of the human red blood cell were investigated. NBDG entry is inhibited by increasing concentrations of d-glucose (K_i = 2 mM). However, NBDG exit is unaffected by d-glucose in red blood cells. Cytochalasin B was found to inhibit both NBDG entry and exit. NBDG accumulates in the red blood cell above the theoretical equilibrium concentration. Accumulation of NBDG is temperature-sensitive and is due to the binding of NBDG to some intracellular substance. The binding of NBDG to purified hemoglobin suggests that accumulation of NBDG by erythrocytes is due to the intracellular binding of NBDG to hemoglobin. NBDG does not accumulate in pink erythrocyte ghosts, while its rate of uptake is still inhibited by d-glucose and cytochalasin B. Although there was no apparent d-glucose inhibition of NBDG exit by intact red blood cells, d-glucose was able to inhibit NBDG exit by pink erythrocyte ghosts. The differing properties of NBDG influx and efflux support the interpretation that the hexose transport system of the human red blood cell appears asymmetric although it may be intrinsically symmetric.     

Vanadate affects glucose metabolism of human erythrocytes

Vanadate causes a rapid breakdown of 2,3-bisphosphoglycerate in intact erythrocytes. This metabolite is nearly stoichiometrically transformed into pyruvate, which changes the cell redox state and enhances the glycolytic flux. The results show that the vanadate effect on 2,3-bisphosphoglycerate, also evident in hemolysates, is attributable to the stimulation of a phosphatase activity of the phosphoglycerate mutase. In agreement with others (J. Carreras, F. Climent, R. Bartrons and G. Pons (1982) Biochim. Biophys. Acta705, 238–242), vanadate is thought to destabilize the phosphoryl form of this enzyme which shows competitive inhibition between the ion and 2,3-bisphosphoglycerate in the mutase reaction. A competitive inhibition between vanadate and glucose 1,6-bisphosphate is also found for phosphoglucomutase, without evidence for phosphatase activity toward the bisphosphate cofactor.     

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.     

Effects of pressure on glucose transport in human erythrocytes.

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

Thiol-fatty acylation of the glucose transport protein of human erythrocytes

Incubation of intact human erythrocytes with [3H]palmitate labeled a protein with electrophoretic characteristics of the glucose transporter. This labeling occurred via a thioester linkage, since it was unaffected by organic solvent extraction, but was substantially removed as the hydroxamate upon treatment with neutral hydroxylamine. Immunoprecipitation of the labeled protein with a monoclonal antibody to the glucose transporter confirmed its identity.     

Quantitation of sulfhydryl groups on erythrocytes in polycythemia vera

Summary The number of sulfhydryl groups on the surface of intact erythrocytes from patients with polycythemia vera is significantly less (34%) than those from control subjects. Sulfhydryl groups were measured indirectly by reversibly blocking-SH groups with the thiol reagent, 6,6-dithiodinicotinic acid, which forms stable mixed disulfides on the surface of erythrocytes. Glutathione was used to break the disulfides and release thione into the supernatant. Thione was then quantitated spectrophotometrically.Supported by NIH Biomedical Research Development Grant 1-S08-RR-09171-01     

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.     

Cholinergic stimulation of glucose transport in human erythrocytes

The effects of cholinergic stimulation on glucose equilibrium exchange rate have been studied in human erythrocytes. Carbamylcholine increases the V of equilibrium exchange by 20% but has no significant effect on K_m. The cholinergic effect is abolished by the muscarinic antagonist atropine or by alterations in intracellular calcium concentrations induced by the calcium ionophore A23187.