Glucose Carrier Of Erythrocytes Research Articles

Decrease of Red Cell Membrane Fluidity and -SH Groups Due to Hyperglycemic Conditions Is Counteracted by α-Lipoic Acid1

Human red cell membranes (ghosts) were treated by 5 min of incubation with fasting or hypo- and hyperglycemic concentrations ofD-glucose. This simulation of nondiabetic or diabetic conditions revealed an influence on membrane fluidity and on protein -SH reactivity. Protein -SH groups, measured with Ellman's reagent, generally behave in the same way as membrane fluidity determined with diphenylhexatriene. Maximal values were obtained with 5 mM D-glucose, whereas decrease was observed above 10 mM D-glucose. Addition of α-lipoic acid (4 nmol/mg protein) resulted in a significant increase in membrane fluidity and titratable -SH groups at glucose concentrations of 10 mMand above. Dithiothreitol diminished titrable -SH groups and did not restore membrane fluidity. 2-Mercaptopropionylglycine was only effective in restoration of -SH groups. By contrast toD-glucose, other sugars such asL-glucose,D-fructose, or sucrose revealed no comparable changes on membrane fluidity and titratable membrane -SH groups between concentrations of 5 and 10 mM. The hyperglycemic effects ofD-glucose were corroborated with isolated, reconstituted membrane proteins and erythrocyte glucose carrier, indicating that, in general, the observed divergent biochemical/biophysical changes of the red cell membrane are influenced by the glucose transport protein GluT1. The naturalR-form and theS-form of α-lipoic acid were compared with racemicR-/S-forms for their efficiencies in alterations of red cell membrane fluidity. Decreased fluidities in presence of 10 mMglucose were found to be influenced in differentiated ways: theS-form was highly active in increasing fluidity at 4 nmol/mg and increasingly less active up to 20 nmol/mg protein. By contrast theR-form of lipoic acid was moderately efficient in increasing fluidity through a larger concentration range between 4 and 80 nmol/mg protein.

Monitoring conformational change in the human erythrocyte glucose carrier: use of a fluorescent probe attached to an exofacial carrier sulfhydryl.

Several fluorescent sulfhydryl reagents were tested as probes for assessing substrate-induced conformational change of the human erythrocyte glucose carrier. Of these, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (Mal-ANS) inhibited 3-O-methylglucose transport most strongly and specifically labeled a previously characterized exofacial sulfhydryl on the glucose carrier. Analysis of equilibrium cytochalasin B binding in cells treated with Mal-ANS suggested that the inhibition of transport was due to a partial channel-blocking effect, and not to competition for the substrate binding site or to hindrance of carrier conformational change. In purified glucose carrier prepared from cells labeled on the exofacial sulfhydryl with Mal-ANS, a blue shift in the peak of fluorescence indicated that the fluorophore was in a relatively hydrophobic environment. Mal-ANS fluorescence in such preparations was quenched by ligands with affinity for the outward-facing carrier (ethylidene glucose, D-glucose, and maltose), but not by inhibitors considered to bind to the inward-facing carrier conformation (cytochalasin B or phenyl beta-D-glucoside). The effect of ethylidene glucose appeared to be related to an interaction with the glucose carrier, since the concentration dependence of ethylidene glucose-induced quench correlated well with the ability of the sugar analog to inhibit cytochalasin B binding to intact cells. The hydrophilic quenchers iodide and acrylamide decreased carrier-bound Mal-ANS fluorescence, resulting in downward-curving Stern-Volmer plots. Whereas ethylidene glucose enhanced iodide-induced quench, it had no effect on that of acrylamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of glucose carrier in human erythrocyte water permeability.

Although the transport properties of human erythrocyte water channels have been well characterized, the identity of the protein(s) mediating water flow remains unclear. Recent evidence that glucose carriers can conduct water raised the possibility that the glucose carrier, which is abundant in human erythrocytes, is the water channel. To test this possibility, water permeabilities and glucose fluxes were measured in large unilamellar vesicles (LUV) containing human erythrocyte lipid alone (lipid LUV), reconstituted purified human erythrocyte glucose carrier (Glut1 LUV), or reconstituted glucose carrier in the presence of other human erythrocyte ghost proteins (ghost LUV). In glucose and ghost LUV, glucose carriers were present at 25% of the density of native erythrocytes, were oriented randomly in the bilayer, and exhibited characteristic inhibition of glucose flux when exposed to cytochalasin B. Osmotic water permeability (Pf, in centimeters per second; n = 4) averaged 0.0012 +/- 0.00033 in lipid LUV, 0.0032 +/- 0.0015 in Glut1 LUV, and 0.006 +/- 0.0014 in ghost LUV. Activation energies of water flow for the three preparations ranged between 10 and 13 kcal/mol; p-(chloromercuri)benzenesulfonate (pCMBS), an organic mercurial inhibitor of erythrocyte water channels, and cytochalasin B did not alter Pf. These results indicate that reconstitution of glucose carriers at high density increases water permeability but does not result in water channel activity. However, because the turnover number of reconstituted carriers is reduced from that of native carriers, experiments were also performed on erythrocyte ghosts with intact water channel function. In ghosts, Pf averaged 0.038 +/- 0.013 (n = 9), while the activation energy for water flow averaged 3.0 +/- 0.3 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of a reactive exofacial sulfhydryl on the glucose carrier of human erythrocytes

Tryptic digestion studies of the human erythrocyte glucose carrier have shown that a reactive and transport-sensitive exofacial sulfhydryl is located in the carboxy-terminal half of the molecule, corresponding to Cys347, Cys421, or Cys429. In the present studies, the erythrocyte glucose carrier labeled on the exofacial sulfhydryl with bis(maleimidomethyl) ether-L-[35S]cysteine was chemically cleaved, either at tryptophans by N-bromosuccinimide or at nonalkylated cysteines by 2-nitro-5-thiocyanobenzoic acid. The resulting fragments were separated by linear gradient polyacrylamide gel electrophoresis, and the labeled fragments were identified by their apparent molecular weight, and by immunoblotting with antibodies to specific regions of the carrier protein. All of the labeled fragments were recognized by an antibody to the carboxy terminus of the carrier, but not by an antibody to a cytoplasmic loop on the C-terminal half of the carrier. The labeled exofacial sulfhydryl was assigned to Cys429, since this is the only residue of the three possibilities which is beyond the expected cleavage sites of the two reagents in the carrier sequence. These results concur with the predictions of hydropathy analysis and will be relevant for studies of how modification of this sulfhydryl affects carrier function, particularly since several other known carrier isoforms lack a corresponding cysteine.

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.

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 μM (EC 50), 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 (EC 50 = 63 μM) and protein-depleted ghosts (EC 50 = 61 μM), (2) inhibited cytochalasin B photolabeling of the band 4.5 carrier region in electrophoretic gels of protein-depleted ghosts (EC 50 = 50 μM), 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 occured by activation of the α,β-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 M r = 18000–20000 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.

Specificity of the glucose channel formed by protein D1 of Pseudomonas aeruginosa

Protein D1 from the outer membrane of Pseudomonas aeruginosa was purified and reconstituted into proteoliposomes. Many small molecules were shown to diffuse through the D1 channel in the proteoliposomes, and the permeation rates of d-glucose, l-glucose, d-xylose, and l-xylose were much higher than expected for their size. This finding and the permeation rates of various glucose analogs suggest that, although the channel has a specific recognition site for glucose, it functions in a manner very different from that of the glucose carrier of erythrocytes.

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

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: Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Asymmetric binding of steroids to internal and external sites in the glucose carrier of erythrocytes

Steroids inhibit glucose transport in erythrocytes by binding to sites in the carrier which are exposed on both the outer and inner surfaces of the cell membrane. Some steroids are bound almost exclusively at inner sites (androstendione and androstandione), while others are bound about as firmly on one side as the other (corticosterone). Still others exhibit a moderate preference for the internal site (deoxycorticosterone). The inhibition is in all cases competitive with respect to a substrate which is bound at the same surface of the membrane as the inhibitor. However, in experiments on substrate entry, internally bound inhibitors act in an apparently non-competitive fashion, as expected if the carrier model is valid. This behaviour explains the appearance of competitive, non-competitive and mixed inhibitions with different steroids (Lacko, L., Wittke, B. and Geck, P. (1975) J. Cell Physiol. 86, 673–680).