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. With 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.

Modulation of red blood cell sugar transport by lyso-lipid.

The in vitro presentation to red blood cells of specific lysolipids in amounts comparable to lysolipid levels in serum is shown to markedly influence protein-mediated glucose transport. Lysolipids were introduced exogenously into cell membranes by incubating erythrocytes in buffer containing varying concentrations of lysolipid (under 3.2 microM). The transport-modulating potency of the lysolipids was found to be dependent both on headgroup and hydrocarbon chain. MPL (monopalmitoyl lecithin, L-alpha-lysopalmitoylphosphatidylcholine) had the greatest influence on sugar transport. 15 min incubation of red cells in MPL suspensions sufficed for 99% association of the lysolipid with the cell membranes. This association correlated with altered red-cell sugar transport. At MPL/bilayer lipid molar ratios as low as 0.03%, MPL was found to act as a reversible, hyperbolic, mixed-type inhibitor of exchange D-glucose exit (both Km(app) and Vmax for transport are reduced). Dissociation of MPL from the membrane results in the recovery of original transport activity. MPL at 1.5.10(-17) mol MPL/red cell was found to reduce Ki(app) for D-glucose inhibition of cytochalasin B binding to the glucose carrier protein in red cell ghost membranes. Our findings demonstrate that red-cell membrane-exogenous lysolipid associations can significantly modify protein mediated sugar transport. The simplest explanation of our findings is a direct interaction of lysolipid with the transport protein.

Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system.

The cytochalasin B binding component of the human erythrocyte monosaccharide transport system has been purified. The preparation appears to contain one major protein with an apparent polypeptide chain molecular weight of 55,000 and about 0.4 binding sites per chain. Cytochalasin B binds to the reconstituted preparation with a dissociation constant of 1.3.10(-7) M, a value which is similar to that reported for the transport system in the intact erythrocyte.

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.

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

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

Familial hyperinsulinemia complicated by extreme insulin resistance during pregnancy: a probable postreceptor defect.

Detailed studies of a family with hyperinsulinemia are reported. The index patient, a 30-yr-old woman with polycystic ovary syndrome, presented with gestational diabetes which was completely resistant to insulin in the presence of severe endogenous hyperinsulinemia. Sensitivity to insulin was regained after delivery. Therapy with cyproterone acetate and ethinyl estradiol for hirsutism exacerbated the hyperinsulinemia toward the levels occurring in pregnancy, with a concomitant deterioration of glucose tolerance. Five other members of her family also were found to have hyperinsulinemia together with high concentrations of circulating C-peptide. Antibodies to insulin and to insulin receptors were not detected, insulin antagonists were not increased, and insulin degradation in the circulation was normal. Insulin extracted from the patient's serum was identical to normal insulin by the criteria of Sephadex chromatography, placental membrane insulin receptor binding, and stimulation of 2-deoxyglucose uptake in isolated rat adipocytes. Although [125I]insulin binding to erythrocytes of all family members and to the patient's placental membranes was markedly reduced, binding to fibroblast cultures from the patient was normal. Insulin-stimulated glucose transport in these fibroblasts also was normal, but there was a mild (20%) reduction in the concentration of cytochalasin B-binding sites in erythrocyte ghosts. Insulin resistance in this family may be due to a partial defect distal to the insulin receptor. This is asymptomatic unless metabolic stresses (pregnancy or steroid administration) are superimposed.

Effects of ATP depletion on the mechanism of hexose transport in intact human erythrocytes.

Depletion of ATP is known to inhibit glucose transport in human erythrocytes, but the kinetic mechanism of this effect is controversial. Selective ATP depletion of human erythrocytes by 10 micrograms/ml A23187 in the presence of extracellular calcium inhibited 3-O-methylglucose influx noncompetitively and efflux competitively. ATP depletion also decreased the ability of either equilibrated 3-O-methylglucose or extracellular maltose to inhibit cytochalasin B binding in intact cells, whereas neither total high-affinity cytochalasin B binding nor its Kd was affected. Under the one-site model of hexose transport these data indicate that ATP depletion decreases both the affinity of the inward-facing glucose carrier for substrate and its ability to reorient outwardly in intact cells.

Direct effects of biguanides on glucose utilization in vitro.

The effect of the biguanides metformin and phenformin on glucose utilization in isolated cells was studied with IM-9 human lymphocytes. Both agents stimulated glucose consumption from the incubation media. Detectable effects of metformin were seen at 33 mumol/L and detectable effects of phenformin were seen at 1.7 mumol/L. Both agents, at similar concentrations, also stimulated [3H] 2-deoxy-D-glucose uptake. Studies with phenformin indicated that biguanides increase the Vmax of uptake without changing the Km. In contrast to the biguanides, IM-9 cells insulin did not influence either glucose consumption or [3H] 2-deoxy-D-glucose uptake. These data provide evidence, therefore, that biguanides may directly influence the cellular utilization of glucose.

Quench-flow analysis reveals multiple phases of GluT1-mediated sugar transport.

Standard models for carrier-mediated nonelectrolyte transport across cell membranes do not explain sugar uptake by human red blood cells. This means that either (1) the models for sugar transport are incorrect or (2) measurements of sugar transport are flawed. Most measurements of red cell sugar transport have been made over intervals of 10 s or greater, a range which may be too long to measure transport accurately. In the present study, we examine the time course of sugar uptake over intervals as short as 5 ms to periods as long as 8 h. Using conditions where transport by a uniform population of cells is expected to be monophasic (use of subsaturating concentrations of a nonmetabolizable but transported sugar, 3-O-methylglucose), our studies demonstrate that red cell sugar uptake is comprised of three sequential, protein-mediated events (rapid, fast, and slow). The rapid phase is more strongly temperature-dependent than the fast and slow phases. All three phases are inhibited by extracellular (maltose or phloretin) or intracellular (cytochalasin B) sugar-transport inhibitors. The rate constant for the rapid phase of uptake is independent of the 3-O-methylglucose concentration. The magnitude (moles of sugar associated with cells) of the rapid phase increases in a saturable manner with [3-O-methylglucose] and is similar to (1) the amount of sugar that is retained by red cell membrane proteins upon addition of cytochalasin B and phloretin and (2) the d-glucose inhibitable cytochalasin B binding capacity of red cell membranes. These results are consistent with the hypothesis that previous studies have both under- and overestimated the rate of erythrocyte sugar transport. These data support a transport mechanism in which newly bound sugars are transiently sequestered within the translocation pathway where they become inaccessible to extra- and intracellular water.

Binding of cytochalasin B to human erythrocyte glucose transporter.

Cytochalasin B, a potent inhibitor of D-glucose transport systems, binds to the glucose transporter purified from human erythrocytes as described previously [Kasahara, M., & Hinkle, P. C. (1977) J. Biol. Chem. 252, 7384]. The transporter binds 9.2 +/- 1.3 nmol of cytochalasin B/mg of protein with a dissociation constant of 0.18 microM. The binding is competitively inhibited by D-glucose (Ki = 43 mM). Phloretin, diethylstilbestrol, maltose, 6-O-propyl-D-galactose, propyl beta-D-glucopyranoside, and dithiothreitol were also linear competitive inhibitors of cytochalasin B binding. The propyl sugars have been shown to inhibit transport from either the plasma or cytoplasma side of the membrane, respectively. The binding of cytochalasin B to the isolated transporter was inhibited by both propyl sugars.

Characterisation of monoclonal antibodies which specifically recognise the human erythrocyte glucose transport protein.

Two monoclonal antibodies (mabs) of subclass IgG1 have been raised against the human erythrocyte glucose transport protein. The mabs bound to the purified glucose transporter in both its membrane-bound and detergent-solubilised forms. However, they exhibited little or no binding to the detergent-solubilised nucleoside transport protein, which is present as a minor contaminant in the glucose transport protein preparation. Both mabs inhibited the binding of cytochalasin B to the glucose transport protein, reducing the affinity of this binding by greater than 2-fold. Each mab labelled the transporter polypeptide on Western blots both before and after treatment of the protein with endoglycosidase F, indicating that the epitopes recognised were located on the protein moiety of the glycoprotein. However, the mabs did not bind to the large fragments produced by tryptic or chymotryptic digestion of the native protein, although both mabs were shown to bind to sites on the cytoplasmic surface of the erythrocyte membrane.

Equilibria and kinetics of ligand binding to the human erythrocyte glucose transporter. Evidence for an alternating conformation model for transport.

Cytochalasin B (CB), n-propyl beta-D-glucopyranoside (PG), and 4,6-O-ethylidene-D-glucose (EG) are known to bind asymmetrically to the human erythrocyte glucose transporter. The first two compounds bind to the inner (cytoplasmic) surface of the transporter, while the latter binds to the outer surface. Equilibrium measurements of the inhibition of CB binding to the glucose transporter reported herein indicate that the ternary complexes of CB transporter with EG, PG, or D-glucose are not formed. Moreover, measurements of CB binding in the presence of both EG and PG or in the presence of high concentrations of D-glucose show that a ternary complex of transporter and sugars bound simultaneously on both sides of the membrane probably does not occur. Finally, the kinetics of dissociation of radiolabeled CB from the transporter in the presence of CB, glucose, PG, and EG have been determined. With the exception of the case of EG, the kinetics fit a simple scheme of rate-limiting unimolecular dissociation, and in no instance do they suggest the existence of a ternary complex of sugar, CB, and transporter. These data are consistent with a model for transport in which the substrate binding site exists alternately at the cytoplasmic and external faces of the membrane, as the result of protein conformational change.

Structure of cytochalasins and cytochalasin B binding sites in human erythrocyte membranes.

Twenty cytochalasins were tested for binding to and for inhibition of glucose transport in human erythrocyte membrane. In this membrane three cytochalasin B (CB) binding sites have been identified. All but three of the cytochalasins bind at site II. On the other hand, only nine of them, which are structurally closely related, bind at site I and inhibit glucose transport. For site I (and site III) binding and glucose transport inhibitory activities (a) the macrocyclic ring in the cytochalasin molecule must be at least 13-membered, (b) the nature of the aromatic ring at C-10 is not important, (c) the C-20-C-23 region makes a major contribution, and (d) the C-5-C-7 segment has a relatively minor influence. These findings do not support a proposed mechanism which involves 24, C-23, C-20, and C-1 oxygen atoms for interaction of CB with glucose carrier. The structural requirements for site II activity are less stringent. The size and the structure of the macrocyclic ring and the nature of the aromatic residue at C-10 modulate this activity only slightly, if at all. Modifications in the C-5-C-7 region of the molecule, however, result in substantial changes in this activity.

Cytochalasin B binding proteins in human erythrocyte membranes. Modulation of glucose sensitivity by site interaction and partial solubilization of binding activities.

We have previously described three different cytochalasin B binding sites in human erythrocyte membranes, a D-glucose-sensitive site (Site I), a cytochalasin E-sensitive site (Site II), and a site (Site III) insensitive to both D-glucose and cytochalasin E. Ligand bindings to each of these sites were considered to be independent (Jung, C., and Rampal, A. (1977) J. Biol. Chem. 252, 5456-5463). However, we have obtained subsequently the following evidence which indicated that an interaction occurs between Sites II and III, and this modulates sensitivity of Site III to the sugar. The displacement of cytochalasin E greatly exceeds the sum of their independent displacements. This ghosts extracted with EDTA or 2,3-dimethylmaleic anhydride at low ionic strength lack Site II activity but retain Site I and III activities, and both of these activities are displaceable by D-glucose alone. This indicated that the removal of Site II from the membrane confers glucose sensitivity to Site III. These observations are consistent with a model that Sites II and III in the membrane exist in a close association through which unliganded Site II maintains the glucose insensitivity of Site III, and once site II is liganded or removed by extraction this association is disrupted and Site III becomes glucose-sensitive. The ghosts extracted with Triton X-100 retain a cytochalasin B binding activity similar to that of site II (Kd = 1.8 X 10(-7) M, cytochalasin E-sensitive, glucose-insensitive), whereas a binding activity similar to that of Site I (Kd = 4 X 10(-7) M, cytochalasin E-insensitive, glucose-sensitive) is recovered in the Triton extract. A cytochalasin B binding activity similar to that of Site II is solubilized by EDTA at low ionic strength.

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.

Reduction of red cell glucose transporter intrinsic activity in diabetes running.

The function of the red blood cell glucose transporter was compared in samples from subjects with and without diabetes. Activity of the glucose transporting protein (GLUT-1) was measured by determining the first order rate constant for uptake of sorbose, a sugar transported by GLUT-1. Red cells were isolated from 13 patients with diabetes and 9 patients without diabetes and were washed free of intracellular glucose. The uptake rate constant was calculated from measurements of sorbose uptake at 0, 1, 2, 5 and 90 minutes at 37 degrees C. The rate constant was significantly decreased in cells isolated from patients with diabetes (0.242 vs 0.303 min-1 in non-diabetic subjects, p < 0.005). The number of GLUT-1 present per mg of membrane protein and clinical parameters such as weight, age, serum cholesterol and urea nitrogen were not significantly different between the groups. The rate constant per pmol of GLUT-1 was significantly decreased in the diabetic subjects. The relationship between diabetes control and the rate constant was not linear and there was no relationship between the calculated intrinsic activity and the HA1c. Because red cell GLUT-1 are not translocated and red cells do not synthesize new proteins, these data suggest that the intrinsic function of the glucose transporter from red cells of patients with diabetes is diminished. This may be due to alterations in the transporter or its membrane environment.

Interaction among anion, cation and glucose transport proteins in the human red cell.

The time course of binding of the fluorescent stilbene anion exchange inhibitor. DBDS (4.4'-dibenzamido-2.2'-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant. tau DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID50 (0.3 +/- 0.1 microM) for H2-DIDS (4.4'-diisothiocyano-2.2'-dihydrostilbene disulfonate) inhibition of tau DBDS is virtually the same as the ID50 (0.47 +/- 0.04 microM) for H2-DIDS inhibition of red cell Cl- flux, thus relating tau DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in tau DBDS, which can be reversed with intracellular, but not extracellular, D-glucose, ID50 for cytochalasin B modulation of tau DBDS is 0.1 +/- 0.2 microM in good agreement with KD = 0.06 +/- 0.005 microM for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 microM), the specific inhibitor of red cell Na+,K+-ATPase, increases red cell Cl- exchange flux in red cells by a factor of about two. This interaction indicates that the Na+,K+-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport protein complex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.