The effect of ionizing radiation on tryptophan fluorescence of thymocyte and erythrocyte plasma membranes

The effect of ionizing radiation on tryptophan fluorescence of thymocyte and erythrocyte plasma membrane preparations was studied. The intensity of tryptophan fluorescence decreased after applying radiation doses up to 15 Gy. The radiosensitivity of thymocyte membranes appeared to be higher than that of the erythrocyte ghosts. Tryptophan radiolysis did not significantly contribute to the effects of radiation. The fraction of tryptophan residues accessible for quenching by I- decreased from 0.87 in the untreated membranes to 0.63 and 0.49 in membranes after doses of 10 and 250 Gy, respectively. The effective quenching constant and the tryptophan fluorescence polarization increased after irradiation. The mechanisms producing these radiation-induced changes are discussed.     

Hyperglycaemia alters the physico-chemical properties of proteins in erythrocyte membranes of diabetic patients.

1. The dynamic properties of erythrocyte membranes in diabetic children and of control erythrocyte membranes subjected to in vitro glycation have been investigated by means of fluorescence quenching of membrane tryptophan residues and ESR spectroscopy. 2. The apparent distance separating the membrane protein tryptophan and the bound 1-anilino-8-naphthalenesulphonate (ANS) molecules was decreased in erythrocyte membranes from children with diabetes. This resulted in a significant increase of the maximum energy transfer efficiency in diabetic membranes. 3. The relevant alterations occurred in the above parameters due to the in vitro nonenzymatic glycosylation of control membranes. 4. These changes were accompanied by the decreased hw/hs parameter of MSL and the increased relative rotational correlation time (tau c) of ISL in diabetic membranes and in the membranes subjected to in vitro glycation. 5. The results suggest that the conformational changes in membrane proteins may occur at both the intrinsic and exposed thiol groups. 6. Both the in vivo and the in vitro data indicate that nonenzymatic glycosylation of membrane proteins may be the major factor attributable to the alterations in the dynamic properties of erythrocyte membrane in diabetic state.     

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.     

Quantitation of lysine-bound glucose of normal and diabetic erythrocyte membranes by HPLC analysis of furosine [ epsilon-N(L-furoylmethyl)-L-lysine]

Non-enzymatic glycosylation of erythrocyte membranes was studied using a non-radioactive and sensitive procedure for specific quantitation of lysine-bound glucose in proteins. About 2 nmol lysine-bound glucose/mg protein were found in ghosts from normal erythrocytes, and this value was about doubled in diabetic patients. In vitro incubation of normal ghosts with glucose gave rise to levels of lysine-bound glucose similar to those found in diabetics. There was a linear correlation between the amount of lysine-bound glucose of total hemoglobin and of membrane proteins. Membrane glycosylation also depended on the age of erythrocytes displaying significantly higher values in old cell populations.     

Sendai Virus Fusion Activity as Modulated by Target Membrane Components

We have studied the differences between erythrocytes and erythrocyte ghosts as target membranes for the study of Sendai virus fusion activity. Fusion was monitored continuously by fluorescence dequenching of R18-labeled virus. Experiments were carried out either with or without virus/target membrane prebinding. When Sendai virus was added directly to a erythrocyte/erythrocyte ghost suspension, fusion was always lower than that obtained when experiments were carried out with virus already bound to the erythrocyte/erythrocyte ghost in the cold, since with virus prebinding fusion can be triggered more rapidly. Although virus binding to both erythrocytes and erythrocyte ghosts was similar, fusion activity was much more pronounced when erythrocyte ghosts were used as target membranes. These observations indicate that intact erythrocytes and erythrocyte ghosts are not equivalent as target membranes for the study of Sendai virus fusion activity. Fusion of Sendai virus with both target membranes was inhibited when erythrocytes or erythrocyte ghosts were pretreated with proteinase K, suggesting a role of target membrane proteins in this process. Treatment of both target membranes with neuraminidase, which removes sialic acid residues (the biological receptors for Sendai virus) greatly reduced viral binding. Interestingly, this treatment had no significant effect on the fusion reaction itself.     

Transmembrane distribution of sterol in the human erythrocyte

The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.     

Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.     

Evidence that inhibitors of anion exchange induce a transmembrane conformational change in band 3

The transport inhibitor, eosin 5-maleimide, reacts specifically at an external site on the membrane-bound domain of the anion exchange protein, Band 3, in the human erythrocyte membrane. The fluorescence of eosin-labeled resealed ghosts or intact cells was found to be resistant to quenching by CsCl, whereas the fluorescence of labeled inside-out vesicles was quenched by about 27% at saturating CsCl concentrations. Since both Cs+ and eosin maleimide were found to be impermeable to the red cell membrane and the vesicles were sealed, these results indicate that after binding of the eosin maleimide at the external transport site of Band 3, the inhibitor becomes exposed to ions on the cytoplasmic surface. The lifetime of the bound eosin maleimide was determined to be 3 ns both in the absence and presence of CsCl, suggesting that quenching is by a static rather than a dynamic (collisional) mechanism. Intrinsic tryptophan fluorescence of erythrocyte membranes was also investigated using anion transport inhibitors which do not appreciably absorb light at 335 nm. Eosin maleimide caused a 25% quenching and 4,4'-dibenzamidodihydrostilbene-2,2'-disulfonate) caused a 7% quenching of tryptophan fluorescence. Covalent labeling of red cells by either eosin maleimide or BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) caused an increase in the susceptibility of membrane tryptophan fluorescence to quenching by CsCl. The quenching constant was similar to that for the quenching of eosin fluorescence and was unperturbed by the presence of 0.5 M KCl. Neither NaCl nor Na citrate produced a large change in the relative magnitude of the tryptophan emission. The tryptophan residues that can be quenched by CsCl appear to be different from those quenched by eosin or BIDS and are possibly located on the cytoplasmic domain of Band 3. The results suggest that a conformational change in the Band 3 protein accompanies the binding of certain anion transport inhibitors to the external transport site of Band 3 and that the inhibitors become exposed on the cytoplasmic side of the red cell membrane.     

Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites.

The human erythrocyte sugar transporter is thought to function either as a simple carrier (sugar import and sugar export sites are presented sequentially) or as a fixed-site carrier (sugar import and sugar export sites are presented simultaneously). The present study examines each hypothesis by analysis of the rapid kinetics of reversible cytochalasin B binding to the sugar export site in the presence and absence of sugars that bind to the sugar import site. Cytochalasin B binding to the purified, human erythrocyte glucose transport protein (GLUT1) induces quenching of GLUT1 intrinsic tryptophan fluorescence. The time-course of GLUT1 fluorescence quenching reflects a second-order process characterized by simple exponential kinetics. The pseudo-first-order rate constant describing fluorescence decay (kobs) increases linearly with [cytochalasin B] while the extent of fluorescence quenching increases in a saturable manner with [cytochalasin B]. Rate constants for cytochalasin B binding to GLUT1 (k1) and dissociation from the GLUT1.cytochalasin B complex (k-1) are obtained from the relationship: kobs = k-1 + k1[cytochalasin B]. Low concentrations of maltose, D-glucose, 3-O-methylglucose, and other GLUT1 import-site reactive sugars increase k-1(app) and reduce k1(app) for cytochalasin B interaction with GLUT1. Higher sugar concentrations decrease k1(app) further. The simple carrier mechanism predicts that k1(app) alone is modulated by import- and export-site reactive sugars and is thus incompatible with these findings. These results are consistent with a fixed-site carrier mechanism in which GLUT1 simultaneously presents cooperative sugar import and export sites.     

Structural research on the membranes and adjacent layers of erythrocytes during the production of erythrocyte ghosts

Using the polarization microscopy and X-ray crystal analysis it has been shown that the ordered structure is destroyed in membranes and adjacent layers of erythrocytes in the course of erythrocyte ghost preparation. These changes are accompanied by quenching of the birefringence which completely disappears in erythrocytes ghosts. The intensities of reflections in X-ray patterns are essentially decreased and the interlayer distances are changed.     

Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose.

The synthesis of 2-N-[4-(1'-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-++ +yloxy)-2- propylamine (ATB-BMPA) is described. This compound was used as an exofacial probe for the human erythrocyte glucose-transport system. A new method is described for directly estimating the affinity for exofacial ligands which bind to the erythrocyte glucose transporter. By using this equilibrium-binding method, the Ki for ATB-BMPA was found to be 338 +/- 37 microM at 0 degrees C and 368 +/- 59 microM at 20 degrees C. This was similar to the concentration of ATB-BMPA required to half-maximally inhibit D-galactose uptake (Ki = 297 +/- 53 microM). The new photoaffinity reagent labelled the glucose transporter in intact cells but, because of its improved selectivity, was also used to label the glucose transporter in isolated erythrocyte membranes. The ATB-BMPA-labelled glucose transporter was 80% immunoprecipitated by anti-(GLUT1-C-terminal peptide) antibody, which shows that the GLUT1 glucose transporter is the major isoform present in erythrocytes. The labelling of the glucose transporter at its exofacial site, and the adoption of an outward-facing conformation, renders the transport system resistant to thermolysin and trypsin treatment. Trypsin treatment of the unlabelled glucose transporter in erythrocyte membranes produced an 18 kDa fragment which was subsequently labelled by ATB-BMPA, but had low affinity for this exofacial ligand. This suggests that the trypsin-treated transporter adopts an inward-facing conformation. The ability of D-glucose to displace ATB-BMPA from the native transporter and from the 18 kDa trypsin fragment have been compared. The D-glucose concentration which was required to obtain half-maximal inhibition of ATB-BMPA labelling was 6-fold lower for the 18 kDa tryptic fragment.     

Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein

Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.     

Membrane phospholipid asymmetry as a factor in erythrocyte-endothelial cell interactions

The tendency of human erythrocytes to adhere to vascular endothelial cells was assessed as a function of the transbilayer distribution of the phospholipids of the erythrocyte membrane, using erythrocyte ghosts in which transbilayer lipid arrangement was manipulated by varying the conditions under which the ghosts were prepared. By two different assays, ghosts with symmetric lipid bilayers adhered strongly to monolayers of cultured endothelial cells, whereas ghosts with normal asymmetric membranes, like normal erythrocytes, did not. These results provide direct evidence that changes in phospholipid asymmetry can alter the tendency of erythrocytes to adhere to endothelial cells, and therefore imply that transbilayer phospholipid arrangement may influence the behavior of erythrocytes in the circulatory system and may contribute to the formation of microvascular occlusions.     

Does diabetes mellitus affect diphenylhexatriene penetration into erythrocyte membrane ghosts?

Diphenylhexatriene transverse distribution has been studied in normal and diabetic erythrocyte membrane ghosts using fluorescence polarization and fluorescence quenching methods. Acrylamide quenched the fluorescence of diphenylhexatriene according to a dynamic mechanism in agreement with Stern-Volmer equation. Nonlinear least-squares analysis based on quenching results has shown greater accessibility of fluorophore to quencher molecules in diabetic ghosts (37.2 +/- 3.2% in normal vs. 67.5 +/- 6.4% in diabetic membranes). Steady-state fluorescence anisotropy measurements evidenced the lowered membrane lipid fluidity in diabetics (anisotropy values: 0.166 +/- 0.011 in normal subjects vs. 0.193 +/- 0.018 in diabetics). A model mechanism is proposed which attributes the lowered capacity of lipid bilayer in diabetes to the increased ordering and more compact structure of membrane phospholipids. The implications of the results for the resolving of steady-state anisotropy data are discussed.     

Excitation energy transfer from tryptophan residues of peptides and intrinsic proteins to diphenylhexatriene in phospholipid vesicles and biological membranes

An efficient excitation energy transfer from tryptophan residues of intrinsic membrane proteins to an extrinsic fluorescent probe (diphenylhexatriene) has been demonstrated in rat erythrocyte ghosts. To correlate this transfer with the localization of the probe, a model system has been investigated. It consists of peptides containing lysine and tryptophan residues bound to negatively charged phosphatidylserine vesicles. Absorption and fluorescence spectroscopies were used to follow peptide binding and diphenylhexatriene incorporation. Peptide binding is accompanied by a blue shift of the tryptophan fluorescence together with an increase of the quantum yield and of the fluorescence decay time. An experimental Föster critical distance value of 4.0 nm was found for energy transfer from tryptophan residues of peptides to diphenylhexatriene which approaches the range of calculated values (3.1–3.7 nm) using a two-dimensional model. These results demonstrate that efficient energy transfer can occur from tryptophan residues of intrinsic proteins to diphenylhexatriene without any interaction between diphenylhexatriene and proteins in biological membranes.     

Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies

The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55,000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze-thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Water exchange through erythrocyte membranes: p-choloromercuribenzene sulfonate inhibition of water diffusion in ghosts studied by a nuclear magnetic resonance technique

A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, corresponding to a decreased exchange rate. However, the diffusional permeability of ghosts was not significantly different from that of erythrocytes . The changes in water diffusion following exposure to p-chloromercuribenzene sulfonate (PCMBS) have been studied on ghosts suspended in isotonic solutions. It was found that a significant inhibitory effect of PCMBS on water diffusion occurred only after several minutes of incubation at 37°C. No inhibition was noticed after short incubation at 0°C as previously used in some labelling experiments. This indicates the location in the membrane interior of the SH groups involved in water diffusion across human erythrocyte membranes. The nuclear magnetic resonance ( n . m . r . ) method appears as a useful tool for studying changes in water diffusiofl in erythrocyte ghosts with the aim of locating the water channel.     

Anomalous asymmetric kinetics of human red cell hexose transfer: role of cytosolic adenosine 5'-triphosphate

Cytosolic adenosine 5'-triphosphate (ATP) modifies the properties of human red cell sugar transport. This interaction has been examined by analysis of substrate-induced sugar transporter intrinsic fluorescence quenching and by determination of Michaelis and velocity constants for D-glucose transport in red cell ghosts and inside-out vesicles lacking and containing ATP. When excited at 295 nm, human erythrocyte ghosts stripped of peripheral proteins display an emission spectrum characterized by a scattering peak and a single emission peak centered at about 333 nm. Addition of sugar transport substrate or cytochalasin B and phloretin (sugar transport inhibitors) reduces emission peak height by 10% and 5%, respectively. Cytochalasin B induced quenching is a simple saturable phenomenon with an apparent Kd (app Kd) of 60 nM and a capacity of 1.4 nmol of sites/mg of membrane protein. Quenching by D-glucose (and other transported sugars) is characterized by at least two (high and low) app Kd parameters. Inhibitor studies indicate that these sites correspond to sugar efflux and influx sites, respectively, and that both sites can exist simultaneously. ATP induces quenching of stripped ghost fluorescence with half-maximal effects at 20-30 microM ATP. ATP reduces the low app Kd and increases the high app Kd for sugar-induced fluorescence quenching. D-Glucose transport in intact red cells is asymmetric (Km and Vmax for influx less than Km and Vmax for efflux). In addition, two operational Km parameters for efflux are detected in zero- and infinite-trans efflux conditions. Protein-mediated sugar transport in ghosts and inside-out vesicles (IOVs) is symmetric with respect to Km and Vmax for entry and exit, and only one Km for exit is detected. Addition of millimolar levels of ATP to the interior of ghosts or to the exterior of IOVs restores both transport asymmetry and two operational Km parameters for native efflux. A model for red cell hexose transport is proposed in which ATP modifies the catalytic properties of the transport system. This model mimics the behavior of the sugar transport systems of intact cells, ghosts, and inside-out vesicles.     

Melittin-induced alterations in dynamic properties of human red blood cell membranes.

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.     

Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi

Summary The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.