Binding Of Cytochalasin Research Articles

Actin-curcumin interaction: insights into the mechanism of actin polymerization inhibition.

Curcumin, derived from rhizomes of the Curcuma longa plant, is known to possess a wide range of medicinal properties. We have examined the interaction of curcumin with actin and determined their binding and thermodynamic parameters using isothermal titration calorimetry. Curcumin is weakly fluorescent in aqueous solution, and binding to actin enhances fluorescence several fold with a large blue shift in the emission maximum. Curcumin inhibits microfilament formation, which is similar to its role in inhibiting microtubule formation. We synthesized a series of stable curcumin analogues to examine their affinity for actin and their ability to inhibit actin self-assembly. Results show that curcumin is a ligand with two symmetrical halves, each of which possesses no activity individually. Oxazole, pyrazole, and acetyl derivatives are less effective than curcumin at inhibiting actin self-assembly, whereas a benzylidiene derivative is more effective. Cell biology studies suggest that disorganization of the actin network leads to destabilization of filaments in the presence of curcumin. Molecular docking reveals that curcumin binds close to the cytochalasin binding site of actin. Further molecular dynamics studies reveal a possible allosteric effect in which curcumin binding at the "barbed end" of actin is transmitted to the "pointed end", where conformational changes disrupt interactions with the adjacent actin monomer to interrupt filament formation. Finally, the recognition and binding of actin by curcumin is yet another example of its unique ability to target multiple receptors.

Dual action of phenylarsine oxide on the glucose transport activity of GLUT1

An early event in the toxic effects of organic arsenic compounds, such as phenylarsine oxide (PAO), is an inhibition of glucose uptake. Glucose uptake involving the glucose transporter, GLUT4 is inhibited by PAO indicating an importance of vicinal sulfhydryls in insulin-stimulated glucose uptake. However, the data on effects of PAO on GLUT1 are conflicting. This study investigated the effects of PAO on glucose uptake in L929 fibroblast cells, cells, which express only GLUT1. The data presented here reveal a dual effect of PAO. At low concentrations or short exposure times PAO stimulated glucose uptake reaching a peak activation of about 400% at 3 μM. At higher concentrations (40 μM), PAO clearly inhibited glucose uptake. At intermediate concentrations (10 μM), PAO had no effect under basal conditions but completely inhibited activation of glucose uptake by glucose deprivation and partially inhibited methylene blue-stimulated glucose uptake. PAO increased the specific binding of cytochalasin B to GLUT1 suggesting a direct interaction with the transporter. These data are most consistent with PAO interacting with multiple proteins that regulate the activity of this transporter, one of which may be GLUT1 itself. The identity of these proteins will require further investigation.

Endofacial competitive inhibition of the glucose transporter 1 activity by gossypol

Gossypol is a natural disesquiterpene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary cells overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a K(i) value of 20 microM. Likewise, GLUT1 fluorescence was quenched approximately 80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of D-glucose. Gossypol did not alter the affinity of D-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose.

N-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood–brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood–brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (−23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 μM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [ 3H]-3- O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood–brain barrier, possibly via changes in GLUT1 protein expression and activity.

Omega-3 fatty acids and brain energy metabolism: Impact on the expression of glucose transporters and glucose transport activity in endothelial cells in culture

Alterations in cognitive performance and neuronal activity in animals defi cient in n-3 polyunsaturated fatty acids (PUFA) have been linked to the decrease in docosahexaenoic acid (22:6n-3, DHA) level in brain membranes. These alterations could partly result from changes in brain energy metabolism and namely in glucose transport activity. Indeed, we reported that several brain areas of rats raised on a diet defi cient in n-3 PUFAs have below-normal rates of glucose utilization [1]. We postulated that the quantity of glucose transporter GLUT1 in defi cient rats was functionally abnormal as its immunoreactivity was low. We present experimental data that evaluated: 1) the in vivo impact of an n-3 PUFA-defi cient diet on expression of the main glucose transporters of rat cerebral cortex (endothelial 55-kDa GLUT1, astrocytic 45-kDa GLUT1, and neuronal GLUT3); and 2) the in vitro impact of the three main long-chain n-6 and n-3 PUFAs, arachidonic acid (20:4n-6), eicosapentaenoic acid (20:5n-3) and DHA, on the fatty acid composition of membrane phospholipids and glucose uptake in two models of rat brain endothelial cells (primary cultures (RBEC), and immortalized cell line RBE4). In vivo study Rats raised with a n-3 PUFA defi cient diet throughout one generation were compared with rats fed a diet with adequate amounts of n-6 and n-3 PUFA. The fatty acid profi le of brain microvessels was determined in order to identify the alterations in n-6 and n-3 PUFA in endothelial cells compared to their profi le in neural cells from the cerebral cortex. The main glucose transporters were quantifi ed by western immunoblotting and by real time quantitative RT-PCR. Specifi c binding of cytochalasin B to GLUT1 of isolated microvessels was also determined. In vitro study The three PUFA studied were added to the culture medium (15μM) as sodium salts on the fi rst day of culture and until the cells were used. Glucose uptake was measured on monolayers of RBEC and RBE4 cells using the non-metabolisable radiolabelled glucose analogue 3-o-methylglucose ([3H]-3-o-MG). In vivo data showed that the 55-kDa GLUT1 protein quantity decreased by 25% in the n-3 PUFA-defi cient microvessels, as did 45 kDa-GLUT1 in the homogenate (30% decrease). Results from cytochalasin B binding confi rmed these data, showing a lower level of its maximum binding to GLUT1. However, GLUT3 protein quantity did not change as GLUT1 and GLUT3 mRNA level. In vitro data demonstrated that both cerebral endothelial cells had low n-6 (mainly 20:4n-6) and n-3 (mainly 22:6n-3) PUFA concentrations under standard conditions of culture. RBEC and RBE4 cells grown in medium supplemented with 20:4n-6, 20:5n-3 or 22:6n-3 (15 μM, physiological concentrations), avidly incorporated these PUFAs into their membrane phospholipids to approach the contents of cerebral microvessels of control rats (considered as physiological references). However RBE4 cells did not incorporate suffi cient 20:4n-6 compared to physiological concentrations. Supplementation with 20:5n-3 and 22:6n-3 increased the basal glucose transport in RBEC ([3H]-3-O-methylglucose uptake) by 50% and 35% respectively, while 20:4n-6 had no effect. On the other hand, none of the PUFA supplements had a signifi cant effect on glucose uptake by RBE4. We demonstrated that incorporation of n-3 long-chain PUFAs in RBEC membranes acts on glucose transport activity. We suggest that the decreased glucose utilization in the cerebral cortex of n-3 PUFA-defi cient rats is in part due to reduced amounts of the two GLUT1 isoforms, possibly issuing from post-transcriptional down-regulation of GLUT1 protein synthesis. In vitro studies suggest that n-3 PUFA can directly modulate the activity of glucose transport in endothelial cells by altering their membrane phospholipid content.

Nicotinamide is not a substrate of the facilitative hexose transporter GLUT1.

It has been proposed that GLUT1, a membrane protein that transports hexoses and the oxidized form of vitamin C, dehydroascorbic acid, is also a transporter of nicotinamide (Sofue, M., Yoshimura, Y., Nishida, M., and Kawada, J. (1992) Biochem. J. 288, 669-674). To ascertain this, we studied the transport of 2-deoxy-D-glucose, 3-O-methyl-D-glucose, and nicotinamide in human erythrocytes and right-side-out and inside-out erythrocyte membrane vesicles. The transport of nicotinamide was saturable, with a K(M) for influx and efflux of 6.1 and 6.2 mM, respectively. We found that transport of the hexoses was not competed by nicotinamide in both the erythrocytes and the erythrocyte vesicles. Likewise, the transport of nicotinamide was not affected by hexoses or by inhibitors of glucose transport such as cytochalasin B, genistein, and myricetin. On the other hand, nicotinamide blocked the binding of cytochalasin B to human erythrocyte membranes but did so in a noncompetitive manner. Using GLUT1-transfected CHO cells, we demonstrated that increased expression of GLUT1 was paralleled by a corresponding increase in hexose transport but that there were no changes in nicotinamide transport. Moreover, nicotinamide failed to affect the transport of hexoses in both control and GLUT1-transfected CHO cells. Therefore, our results indicates that GLUT1 does not transport nicotinamide, and we propose instead the existence of other systems for the translocation of nicotinamide across cell membranes.

Direct inhibition of the hexose transporter GLUT1 by tyrosine kinase inhibitors.

The facilitative hexose transporter GLUT1 is a multifunctional protein that transports hexoses and dehydroascorbic acid, the oxidized form of vitamin C, and interacts with several molecules structurally unrelated to the transported substrates. Here we analyzed in detail the interaction of GLUT1 with a group of tyrosine kinase inhibitors that include natural products of the family of flavones and isoflavones and synthetic compounds such as the tyrphostins. These compounds inhibited, in a dose-dependent manner, the transport of hexoses and dehydroascorbic acid in human myeloid HL-60 cells, in transfected Chinese hamster ovary cells overexpressing GLUT1, and in normal human erythrocytes, and blocked the glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts. Kinetic analysis of transport data indicated that only tyrosine kinase inhibitors with specificity for ATP binding sites inhibited the transport activity of GLUT1 in a competitive manner. In contrast, those inhibitors that are competitive with tyrosine but not with ATP failed to inhibit hexose uptake or did so in a noncompetitive manner. These results, together with recent evidence demonstrating that GLUT1 is a nucleotide binding protein, support the concept that the inhibitory effect on transport is related to the direct interaction of the inhibitors with GLUT1. We conclude that predicted nucleotide-binding motifs present in GLUT1 are important for the interaction of the tyrosine kinase inhibitors with the transporter and may participate directly in the binding transport of substrates by GLUT1.

Effects of Resalination on Intestinal Glucose Transport in Chickens Adapted to Low Na+ Intakes

In chickens, we have shown that intestinal absorption of glucose via apical SGLT1 and basolateral GLUT2 transport systems is affected by dietary Na+; low-Na+ adapted birds show a dramatic reduction of glucose transporters in both membranes in the rectum, an intermediate response in the ileum and no effects in the jejunum. We have now studied the effect of resalination of low-Na+ adapted chickens on glucose kinetics across SGLT1 (using -methyl-D-glucoside as substrate) and GLUT2 (using D-glucose) and on the specific binding of phlorizin and cytochalasin B, respectively. Twelve-week-old male Leghorn chickens were fed wheat and barley with drinking water containing either 150 mM NaCl (high-Na+ group) or 0. 015 mM (low-Na+ group) for 14 days (serum aldosterone: 242 +/- 6 pg ml-1 in the low-Na+ and 46 +/- 4 pg ml-1 in the high-Na+ group). On day 14, the low-Na+ group was either resalinated with an oral dose of NaCl (9 g (kg body wt)-1) or switched to the high-Na+ condition, for 1 week. Serum aldosterone measured 4 h, 1 day and 7 days after the change in NaCl intake fell to between 30 and 39 pg ml-1. The changes in apical -methyl-D-glucoside and basolateral D-glucose transport observed in the ileum and rectum of low-Na+ adapted animals were completely reversed by resalination within 4 h of NaCl administration to the level of values observed for high-Na+ adapted birds. The good correlation between the -methyl-D-glucoside and D-glucose Vmax and the SGLT1 and GLUT2 density, respectively, supports the view that the increase in apical and basolateral hexose transport found in the ileum and rectum of both groups of resalinated birds is due to an increase in the number of protein transporters. The rapid changes in the number of glucose transporters observed suggest that the target of the regulatory signal(s) involved are the mature enterocytes present in the villi rather than the developing enterocytes in the crypt.

Role of conserved arginine and glutamate residues on the cytosolic surface of glucose transporters for transporter function.

The role of conserved arginine and glutamic acid residues at the cytoplasmic surface of the GLUT4 for transporter function was investigated by site-directed mutagenesis and expression of the constructs in COS-7 cells. Reconstituted glucose transport activity, cytochalasin B binding, and photolabeling with the exofacial label 2-N4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1, 3-bis(d-mannosyloxy)-2-propylamine (ATB-BMPA) was assayed in membranes from transfected cells and corrected for immunoreactivity of expressed transporters. Exchange of Arg 92 (R92L amino acid residues are numbered according to the corresponding residues in the GLUT1) or Arg 333/334 (RR333/4LA) reduced or suppressed transport activity with no or very little effect on photolabeling with ATB-BMPA and cytochalasin B binding. It is suggested that the lack of these residues selectively disturbes the substrate-induced conformational change of the carrier during transport. Exchange of Glu 146 (E146D) or Arg 153 (R153L) markedly reduced transport activity, ATB-BMPA photolabeling, and cytochalasin B binding. Transport activity and ATB-BMPA labeling were abolished in the mutants E329Q, E393D, and R400L, whereas binding of cytochalasin B was normal. Thus, exchange of Glu 329, Glu 393, and Arg 400 appears to arrest the transporter in an inward facing conformation. It is concluded that the conserved arginine and glutamate residues at the cytoplasmic surface of the glucose transporter GLUT4 are essential for its appropriate conformation, and that it is the interaction of charged residues which mediates the oscillation between outward and inward facing states.

Developmental increases in glucose transporter concentration in the sheep placenta.

To explore the molecular basis for the gestational increase in glucose transport capacity of the sheep placenta in vivo, placentas from twin-pregnant ewes at days 75, 110, and 140 postcoitus (n = 6/group) were analyzed for glucose transporter (GT) concentration. Concentration (pmol/mg protein) of D-glucose-inhibitable binding sites, measured by [3H]cytochalasin B binding analysis, increased 3.4 times from mid- to late pregnancy. Concurrently, abundance of GLUT-1 and GLUT-3 protein, measured by immunoblotting with specific polyclonal antibodies, increased 2.3 and 2.9 times, respectively, while abundance of GLUT-1 and GLUT-3 mRNA, measured by Northern blotting, increased 1.8 and 3.9 times, respectively. GLUT-4 protein was undetectable at all stages of pregnancy. Quantitative immunoblotting indicated that GLUT-1 accounted for 86.8 +/- 1.6% at day 75 and 56.1 +/- 4.1% at day 140 of total cytochalasin B binding sites. Thus increases in GT concentration explain much of the gestational increase in glucose transfer capacity observed in vivo. The gestational decline in relative contribution of GLUT-1 to cytochalasin binding, together with the greater developmental increases in GLUT-3 mRNA and protein, further suggests that the relative importance of GLUT-3 increases with gestational age.

Genistein Is a Natural Inhibitor of Hexose and Dehydroascorbic Acid Transport through the Glucose Transporter, GLUT1

Genistein is a dietary-derived plant product that inhibits the activity of protein-tyrosine kinases. We show here that it is a potent inhibitor of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, genistein inhibited the transport of dehydroascorbic acid, deoxyglucose, and methylglucose in a dose-dependent manner. Transport was not affected by daidzein, an inactive genistein analog that does not inhibit protein-tyrosine kinase activity, or by the general protein kinase inhibitor staurosporine. Genistein inhibited the uptake of deoxyglucose and dehydroascorbic acid in Chinese hamster ovary (CHO) cells overexpressing GLUT1 in a similar dose-dependent manner. Genistein also inhibited the uptake of deoxyglucose in human erythrocytes indicating that its effect on glucose transporter function is cell-independent. The inhibitory action of genistein on transport was instantaneous, with no additional effect observed in cells preincubated with it for various periods of time. Genistein did not alter the uptake of leucine by HL-60 cells, indicating that its inhibitory effect was specific for the glucose transporters. The inhibitory effect of genistein was of the competitive type, with a Ki of approximately 12 microM for inhibition of the transport of both methylglucose and deoxyglucose. Binding studies showed that genistein inhibited glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts in a competitive manner, with a Ki of 7 microM. These data indicate that genistein inhibits the transport of dehydroascorbic acid and hexoses by directly interacting with the hexose transporter GLUT1 and interfering with its transport activity, rather than as a consequence of its known ability to inhibit protein-tyrosine kinases. These observations indicate that some of the many effects of genistein on cellular physiology may be related to its ability to disrupt the normal cellular flux of substrates through GLUT1, a hexose transporter universally expressed in cells, and is responsible for the basal uptake of glucose.

The Role of Tryptophans 371 and 395 in the Binding of Antibiotics and the Transport of Sugars by the D-Galactose-H+ Symport Protein (GalP) from Escherichia coli

The interactions between the D-galactose-H+ symporter (GalP) from Escherichia coli and the inhibitory antibiotics, cytochalasin B and forskolin, and the substrates, D-galactose and H+, have been investigated for the wild-type protein and the mutants Trp-371-->Phe and Trp-395-->Phe, so that the roles of these residues in the structure-activity relationship could be assessed. Neither mutation prevented photolabeling by either [4-3H]cytochalasin B or by 3-[125I]iodo-4-azidophenethyl-amido-7-O-succinyldesacetylforskolin ([125I]APS-forskolin). However, measurements of protein fluorescence show that both residues are in structural domains, the conformations of which are perturbed by the binding of cytochalasin B or forskolin. Moreover, both mutations cause a substantial decrease in the affinity of the inward-facing site of the GalP protein for cytochalasin B, 10- and 43-fold, respectively, but have little effect upon the affinity of this site for forskolin, 0.8- and 2.6-fold reductions, respectively. Both these mutations change the equilibrium between the putative outward- (T1) and inward-facing (T2) conformations, so that the inward-facing form is more favored. They also stabilize a different conformational state, "T3-antibiotic," in which the initial interactions between the protein and antibiotics are tightened. Overall, this has the effect of compensating for the reduction in affinity for cytochalasin B, so that the respective overall Kd values are 0.74- and 3.5-fold that of the wild type, while causing a slight increase, 1.5- and 3.2-fold, respectively, in affinity of the mutants for forskolin. The Trp-371-->Phe mutation causes a 15-fold reduction in the affinity of the inward-facing site for D-galactose, suggesting that this residue forms part of the sugar binding site. In contrast, the Trp-395-->Phe mutation has no effect upon the affinity of the inward-facing site for D-galactose. These effects may be related to the reduction in galactose-H+ symport activity only in the Trp-371-->Phe mutant, although it still effects active transport to the same extent as the Trp395-->Phe mutant. However, there is a 10-20-fold increase in the Km values for energized transport of D-galactose for both mutants.

The Glucose Transporter in the Plasma Membrane of the Outer Segments of Bovine Retinal Rods

The facilitated diffusion glucose transporter in the plasma membrane of intact outer segments isolated from bovine retinal rods (ROS) was characterized by measurements of: (1) 14C-labeled 3-O-methylglucose fluxes; and (2) glucose-sensitive binding of 3H-labeled cytochalasin B to ROS membranes. Inhibition of 3-O-methylglucose influx into ROS, inhibition of 3-O-methylglucose efflux from ROS and glucose-sensitive binding of cytochalasin B to ROS showed very similar cytochalasin B inhibition/dissociation constants of 0·9 μM, 1·3 μM and 1·3 μM, respectively. d-glucose inhibited both 14C-labeled 3- O-methylglucose transport and cytochalasin B binding. The above results suggest that D-glucose-sensitive cytochalasin B binding reflects specific binding to the ROS glucose transporter and the density of glucose transporter in the ROS plasma membrane was determined to be 800 μm -2, comparable to relatively abundant ROS plasma membrane proteins such as the cGMP-gated channel and the Na-Ca+K exchanger. Displacement of 3H-labeled cytochalasin B by non-transportable hexoses was used to localize the hexose transporters to the ROS plasma membrane and to examine a simple single-site, alternating conformation model for hexose transport. A comparison between the Glut1 glucose transporters of bovine ROS, bovine erythrocytes and human erythrocytes suggests that kinetic and pharmacological characteristics of glucose transporters cannot be predicted in a simple manner from gene type and species.

Equilibrium and transient kinetic studies of the binding of cytochalasin B to the l‐arabinose–H+ symport protein of Escherichia coli

The kinetics of the binding of cytochalasin B to the L-arabinose-H+ symport protein of Escherichia coli have been investigated, using a strain that over-produces the symport protein in the cytoplasmic membrane. Equilibrium binding studies revealed a single set of binding sites (2.9-8.9 nmol/mg protein) with a Kd of 0.7-1.0 microM at 22 degrees C. It proved possible to follow the transient kinetics of cytochalasin B binding by measuring the changes in the fluorescence of the L-arabinose-H+ symporter upon binding the ligand, by stopped-flow fluorescence spectroscopy. The association and dissociation rate constants thus determined were confirmed by rapid filtration measurements, using [3H]cytochalasin B, yielding values of 4.5-6.5 microM-1.s-1 and 4-5 s-1, respectively, consistent with Kd values obtained by measuring equilibrium binding of [3H]cytochalasin B by dialysis at 22 degrees C. Titration of the protein fluorescence with cytochalasin B yielded a similar binding site concentration and Kd value to those obtained in equilibrium binding studies. All the measurements of binding site concentration are consistent with a stoichiometry of 1 mol cytochalasin B binding sites/mol L-arabinose-H+ symport protein. Inhibition of both the rate and equilibrium binding of cytochalasin B by sugars indicated the following order of substrate binding 5-thio-D-glucose > D-fucose > L-arabinose > 6-deoxy-6-fluoro-D-galactose > D-xylose approximately 6-deoxy-D-glucose > D-galactose > D-glucose > D-ribose. Neither D-arabinose nor L-fucose had any significant inhibitory effect upon cytochalasin B binding.

Rhein inhibits glucose uptake in Ehrlich ascites tumor cells by alteration of membrane-associated functions.

Rhein (RH), 4,5 dihydroxyanthraquinone-2-carboxylic acid, is known to inhibit the glycolysis of neoplastic cells by impairing glucose uptake. In order to establish whether this might be due to a selective interaction of the carrier with the drug or to functional modifications of the cell membrane, the effect of RH on glucose uptake in Ehrlich ascites tumor cells has been investigated. RH strongly inhibits the uptake of both 2-deoxyglucose and 3-O-methylglucose, so the reduced influx therefore cannot be ascribed to an effect on glucose phosphorylation. The inhibition of glucose transport does not depend on a reduction of the number of the carriers as indicated by the inability of the drug to interfere with the synthesis of the transporter. Moreover, the extent of total binding of cytochalasin B, as well as the fact that glucose specificity is not altered, indicate that the intrinsic activity of the glucose carrier is not affected. We therefore conclude that the inhibition of glucose uptake must be ascribed to an interaction of the drug with cell membranes that results in an alteration of membrane-associated functions.

Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG2 (Glut1) glucose transporter inhibit transport activity and targeting to the plasma membrane in Xenopus oocytes.

All 6 tryptophan residues in the human HepG2-type glucose transporter (Glut1) were individually altered by site-directed mutagenesis to investigate the role of these residues in transport function. Tryptophan residues in positions 48, 65, 186, 363, 388, and 412 of Glut1 were changed to either a glycine or leucine residue. Mutant mRNAs were synthesized and injected into Xenopus laevis oocytes. Transporter function as assessed by uptake of 2-deoxy-D-[3H]glucose or transport of 3-O-[3H]methylglucose was decreased in the 388 and 412 mutants but was unaltered in all other mutants. The amount of the mutant transporters expressed in total membrane and plasma membrane fractions was measured using Glut1-specific antibodies. Calculation of the intrinsic transport activity of each of the mutants using these data demonstrated that the reduced transport activity of the 412 mutants was caused entirely by a dramatic decrease in the intrinsic activity of the mutant proteins whereas the reduced activity of the 388 mutants was a result of a decreased level of the protein in oocytes, decreased targeting to the plasma membrane, and a modest decrease in the intrinsic activity. Protease/glycosidase mapping of in vitro translation products indicated that the effects of the 388 and 412 point mutations could not be attributed to a disruption in the ability of the mutant proteins to insert properly into the membrane. The ID50 for cytochalasin B inhibition of 2-deoxyglucose uptake was increased from 5 x 10(-7) M for the wild-type Glut1 to 4 x 10(-6) M in the 388 mutants but was unaltered in the 412 mutants. These observations suggest that 1) Trp-412 may comprise part of a hexose binding site or is involved in maintaining a local tertiary structure critical for transport function; 2) Trp-388 is involved in stabilizing the equilibrium binding of cytochalasin B to the transporter. Trp-388 may therefore lie near a substrate binding site and also appears to participate in stabilization of local tertiary structure important for full catalytic activity and efficient targeting to the Xenopus plasma membrane.

Monoclonal antibodies possibly recognize conformational changes in the human erythrocyte glucose transporter

Two monoclonal antibodies (MAG17 and MAG20) were raised against the human erythrocyte glucose transporter, which was purified on an immunoaffinity column using a polyclonal antibody to the C-terminal peptide (residues 477-492) of the glucose transporter of HepG2 cells. To obtain antibodies which recognize the native glucose transporter integrated in the membrane, hybridomas were screened both by e.l.i.s.a. with purified glucose transporter and by dot-blotting with erythrocyte membranes. The antibodies immunoprecipitated D-glucose-inhibitable [3H]cytochalasin B-photoaffinity-labelled glucose transporters, but did not recognize the transporter on Western blotting. The presence of the C-terminal peptide did not inhibit the binding of these antibodies to the glucose transporter, suggesting that the antibodies recognized sites different from the transporter C-terminus. D-Glucose (0.1-100 microM) inhibited the binding of MAG17 and MAG20 to the transporter by 50%, indicating that the conformation of the epitopes was altered allosterically by D-glucose. Cytochalasin B inhibited the binding of MAG17 to the transporter, but enhanced the binding of MAG20 at low concentrations (less than 0.02 microM). These data suggest that the glucose transporter has high- and low-affinity binding sites for D-glucose and cytochalasin B, and that binding of D-glucose and cytochalasin B induces conformational changes in the transporter. Monoclonal antibodies which recognize the tertiary structure of the glucose transporter can be used for investigating its function and structure when integrated in the membrane.

High-Affinity Binding of Cytochalasin B to the B-End of F-Actin Loses Its Inhibitory Effect on Subunit Exchange When the Bound Nucleotide Is ADP1

We investigated the mode of binding of cytochalasin B (CB) to F-actin in an ADP-solution with and without inorganic phosphate (Pi). In the presence of Pi (20 mM), a filament of F-actin had a single high-affinity CB binding site (Kd = 1.4 nM), just like in the case of an ATP-solution [Kd = 5.0 nM: Suzuki, N. & Mihashi, K. (1991) J. Biochem. 109, 19-23]. But in the absence of Pi, there were two low-affinity (Kd = 200 nM) CB binding sites as well as one high-affinity site (Kd = 1.6 nM). We determined the concentration of CB necessary for half-maximal inhibition of growth or shortening of F-actin (Ki) using of pyrene-labeled actin. We obtained Ki = 80 nM for growth and Ki = 800 nM for shortening in the presence of ATP. The addition of Pi to the ATP-solution reduced Ki for growth to 9 nM. We propose a model explaining these results. In the model, high-affinity CB binding to the terminal subunit dimer can inhibit subunit exchange at the B-end only when the terminal subunits bind ATP or ADP.Pi. When the terminal subunits bind ADP, additional low-affinity CB bindings to the terminal subunits are needed to inhibit the subunit exchange.

Myocardial glucose utilization. Failure of adenosine to alter it and inhibition by the adenosine analogue N6-(L-2-phenylisopropyl)adenosine.

The effects of adenosine and the nonmetabolizable adenosine analogue N6-(L-2-phenylisopropyl)adenosine (PIA) on glucose transport or metabolism were determined in purified myocardial sarcolemmal vesicles, isolated cardiocytes, and perfused hearts. Adenosine (100 microM) did not affect hexose transport in myocytes. Also, adenosine deaminase, added to metabolize adenosine to inosine, did not alter transport of hexose into myocytes regardless of whether or not insulin was present. In contrast, PIA effectively inhibited 3-O-methyl-D-glucose uptake in myocytes even during insulin stimulation. PIA inhibited D-glucose-specific transport in both rat and bovine cardiac sarcolemmal vesicles (Ki = 26 microM at [D-glucose] = 5 mM). However, insulin did not affect glucose transport in sarcolemmal vesicles, which implies that receptor-coupled processes probably are not intact in this preparation. Thus, inhibition of PIA may not be receptor mediated. Also, PIA inhibited binding of cytochalasin B to bovine cardiac sarcolemmal vesicles, which supports the idea that PIA inhibits glucose flux by binding to the glucose transporter. To determine if adenosine altered glucose metabolism rather than transport, we measured the rate of 3H2O production from metabolism of D-[2-3H]glucose in paced rat hearts ([D-glucose] = 5.5 mM, [pyruvate] = 0.2 mM) perfused with a range of PIA or adenosine concentrations with or without 0.01 microM insulin. Adenosine (0.01-100 microM) in the presence or absence of insulin increased coronary flow but did not change glycolytic rates. Similar results were obtained with PIA (no insulin) rather than adenosine in the perfusate. However, with glucose as the only exogenous substrate, 100 microM PIA inhibited glycolysis by approximately 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-VLDL increases endothelial cell plasma membrane cholesterol.

In this study, the distribution of free cholesterol in cholesterol-loaded endothelial cells was examined. For these studies, cell fractionation methods were used to assess marker enzyme activity and cholesterol distribution. Treatment of rabbit aortic endothelial cells for 3 days with 50 micrograms/ml of beta-very low density lipoprotein (beta-VLDL) or malondialdehyde-low density lipoprotein (MDA-LDL) but not LDL caused a 50-100% increase in total cell unesterified cholesterol. The accumulation of free rather than esterified cholesterol in endothelial cells may be due to the ratio of hydrolysis to esterification, which we have shown in this study to be 10-fold higher in endothelial cells than in smooth muscle cells. This free cholesterol is found in the fractions enriched in plasma membrane markers and, to a lesser extent, in the Golgi-enriched fractions. The amount of cholesterol per mg of protein was increased approximately 50% in these fractions from cells treated for 3 days with 50 micrograms/ml of beta-VLDL. These increases in cholesterol content were reversible upon incubation of cells for 3 days in medium containing 15% fetal bovine serum. Alterations in several membrane functions were also observed in cholesterol-loaded cells. The activity of alkaline phosphatase, an enzyme marker for plasma membranes, was decreased by 25% and an alteration in membrane-associated microfilaments was seen with phalloidin staining. This morphological change in microfilaments was reflected in a decrease in filament ends as shown by cytochalasin binding and occurred without a change in total actin or vinculin. These microfilament changes were reversible.(ABSTRACT TRUNCATED AT 250 WORDS)