Red Blood Cell Ghost Membranes Research Articles

Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellata root extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.

Dipole relaxation in erythrocyte membrane: Involvement of spectrin skeleton

Polarization of spectrin–actin undermembrane skeleton of red blood cell (RBC) plasma membranes was studied by impedance spectroscopy. Relatedly, dielectric spectra of suspensions that contained RBCs of humans, mammals (bovine, horse, dog, cat) and birds (turkey, pigeon, duck), and human RBC ghost membranes were continuously obtained during heating from 20 to 70°C. Data for the complex admittance and capacitance were used to derive the suspension resistance, R, and capacitance, C, as well as the energy loss as a function of temperature. As in previous studies, two irreversible temperature-induced transitions in the human RBC plasma membrane were detected at 49.5°C and at 60.7°C (at low heating rate). The transition at 49.5°C was evident from the abrupt changes in R, and C and the fall in the energy loss, due to dipole relaxation. For the erythrocytes of indicated species the changes in R and C displayed remarkable and similar frequency profiles within the 0.05–13MHz domain. These changes were subdued after cross-linking of membranes by diamide (0.3–1.3mM) and glutaraldehyde (0.1–0.4%) and at the presence of glycerol (10%). Based on the above results and previous reports, the dielectric changes at 49.5°C were related to dipole relaxation and segmental mobility of spectrin cytoskeleton. The results open the possibility for selective dielectric thermolysis of cell cytoskeleton.

One-step direct reconstitution of biomembranes onto cationic organic polymer bead supports

In this study, we addressed the straightforward reconstitution of red blood cell (RBC) membranes on the surface of cationic organic polymer beads. The RBC membrane–bead complex was obtained by the incubation of white, unsealed rat RBC ghost membranes with a nonporous quaternary ammonium-type anion-exchange polymer bead with a 350–550 μm diameter. Confocal microscopic observations using a fluorescence membrane probe revealed that the RBC membranes were reconstituted on the outer surface of the bead without any remarkable structural gaps in the membrane. The absence of activity of two peripheral enzymes that latently reside on the cytoplasmic face of the RBC membranes demonstrated that the orientation of the RBC membranes immobilized on the beads was asymmetric as well as that in the native state. The RBC membrane–polymer bead complex was incubated with a primary antibody that is directed against the amino-terminal extracellular domain of the integral protein glycophorin A (GPA). The resulting complex was further incubated with a fluorescent secondary antibody and then subjected to confocal microscopic observations. Fluorescence resulting in the binding of the secondary antibody was found on the surface of the complex, which indicates that the amino-terminal extracellular domain of GPA is exposed to the surface of the complex. In addition, the anion uptake function of the most abundant integral protein anion-exchanger 1 (AE1) immobilized on the polymer beads was inhibited by pretreatment with its specific inhibitor 4,4′-diisothiocyano-2,2′-stilbene disulfonate, as is observed for the intact RBCs. Based on all these results, the RBC membranes were thought to be reconstituted on the ionic polymer beads by our one-pot procedure while maintaining the orientation and functions of the membrane proteins to some extent.

Interactions of therapeutically active plant flavonols with biological targets : Insights from fluorescence spectroscopic studies

Plant flavonols have attracted much recent attention in view of their novel therapeutic properties (effective against various free radical mediated and other diseases) which make them promising alternatives to conventional therapeutic drugs. However, till date, not much is known, regarding their mode of interactions and binding affinities with relevant biological targets. This article presents perspectives highlighting the usefulness of the exquisitely sensitive 'two color' fluorescence behavior of flavonols (which arise due to highly efficient photoinduced excited state intramolecular proton transfer (ESIPT) reactions) for exploring their interactions, at the molecular level, with biomembranes and proteins, which are the principal biological targets of such drug molecules. In this context, we made exploratory studies on the interactions of some representative therapeutically important flavonols with model and natural membranes (composed of phosphatidylcholine liposomes and red blood cell ghost membranes respectively) and serum albumin proteins. Since the ESIPT process is highly sensitive to external hydrogen bonding perturbation effects, the relative contribution between the two colors is strongly modulated by the local environment of the fluorophore, with dramatic changes in the emission yield, energy, anisotropy (r), lifetime (τ) and related parameters of both ESIPT tautomer and normal fluorescence bands. This provides multiparametric fluorescence probing opportunities, revealing salient details about the nature and location of binding sites as well as quantitative estimates of partition coefficients/binding constants. This promising new approach may be expected to open up new avenues for the 'screening' of the most appropriate flavonoid derivatives, from among numerous structural variants found in nature, as well as the design of relevant synthetic derivatives with improved features.

Change in Expressional Profile of KCl Cotransporter Genes during Human Erythroid Differentiation.

Maintenance of cell volume by regulated cation transport is a fundamental cellular process. The KCl cotransporter (KCC) contributes to red blood cell (RBC) volume regulation, especially in reticulocytes. Erythroid K-Cl cotransport activity is increased in sickle cells (SS RBC) and contribute to SS RBC dehydration, which potentiates sickling. Three cation cotransporter genes, SLC12A4 (KCC1), SLC12A6 (KCC3) and SLC12A7 (KCC4), and several splicing variants, mediate KCC activity in non-neuronal tissues. To determine which KCC isoform(s) predominates in human RBC we examined the quantitative expression patterns of KCC isoforms during erythroid differentiation. We developed a set of real-time RT-QPCR assays specific for KCC1, KCC1b, KCC3a, KCC3b or KCC4, over a 7-log quantitation range and sensitivity of 10 copies per reaction using multiplex amplification of GAPDH as internal controls. In human reticulocytes isolated by magnetic separation using anti-transferrin receptor coated beads, KCC3a mRNA levels were consistently the highest (4–24 fold of KCC1), while KCC4 levels varied from 1 to 7-fold of KCC1 levels (n=8). Message levels for KCC3b were relatively low (20–80% of KCC1), and for KCC1b were negligible (1–2% of KCC1). Substantial variability in the relative levels of KCC1, KCC3a, and KCC4 mRNA was observed among individual samples, but no consistent difference was apparent comparing sickle and normal reticulocytes. Western blot analysis of sickle and normal RBC ghost membranes confirmed the presence of KCC1, KCC3 and KCC4 at the protein level. To evaluate cells at various erythroid differentiation stages, human CD34+ cells were cultured under conditions favoring erythroid differentiation for 26 days. During early in vitro differentiation, KCC1 was the main mRNA species, followed by KCC4, with similar levels of KCC3a and KCC3b. RNA levels for KCC3a and KCC4 increased during maturation and became the most abundant at later stages. KCC1b mRNA remained low, and KCC3b levels decreased during erythroid development. To further define this temporal sequence of KCC expression, cells cultured for 10–17 days were sorted by FACS into four subpopulations (I to IV), characterized by immunostaining for relative expression of CD71 and glycophorin A, with enrichment of pronormoblastsI.basophilic normoblastsII.polychromatophilic normoblastsIII.or orthrochromatic normoblasts and reticulocytesIV.KCC1, KCC3a, KCC3b, and KCC4 were expressed in these populations, with KCC1 as the main KCC species in early precursors (I), KCC3b decreasing >80% during maturation, KCC4 and KCC3a becoming the most abundant in the most differentiated subpopulation (IV).In summary, we identified KCC3a as the predominant KCC isoform in human reticulocytes, followed by KCC4 and KCC1, consistent with the presence of KCC1, KCC3 and KCC4 proteins in RBC membranes. The expression of KCC3a and KCC4 increased during erythriod differentiation in vitro. Variations in relative expression of KCC species are a potential source of inter-individual differences in KCC function for volume regulation. These results also provide the foundation for the possibility of improving SS RBC hydration by reducing gene expression of the major KCC isoforms in RBC.

Membrane Fusion by Single Influenza Hemagglutinin Trimers: KINETIC EVIDENCE FROM IMAGE ANALYSIS OF HEMAGGLUTININ-RECONSTITUTED VESICLES

Influenza hemagglutinin, the receptor-binding and membrane fusion protein of the virus, is a prototypic model for studies of biological membrane fusion in general. To elucidate the minimum number of hemagglutinin trimers needed for fusion, the kinetics of fusion induced by reconstituted vesicles of hemagglutinin was studied by using single-vesicle image analysis. The surface density of hemagglutinin fusion-activity sites on the vesicles was varied, while keeping the surface density of receptor-binding activity sites constant, by co-reconstitution of the fusogenic form of hemagglutinin, HA(1,2), and the non-fusogenic form, HA(0), at various HA(1,2):(HA(1,2) + HA(0)) ratios. The rate of fusion between the hemagglutinin vesicles containing a fluorescent lipid probe, octadecylrhodamine B, and red blood cell ghost membranes was estimated from the time distribution of fusion events of single vesicles observed by fluorescence microscopy. The best fit of a log-log plot of fusion rate versus the surface density of HA(1,2) exhibited a slope of 0.85, strongly supporting the hypothesis that single hemagglutinin trimers are sufficient for fusion. When only HA(1,2) (without HA(0)) was reconstituted on vesicles, the dependence of fusion rate on the surface density of HA(1,2) was distinct from that for the HA(1,2)-HA(0) co-reconstitution. The latter result suggested interference with fusion activity by hemagglutinin-receptor binding, without having to assume a fusion mechanism involving multiple hemagglutinin trimers.

The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells.

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca2+-activated K+ channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca2+, in the presence but not the absence of external Ko+ and remains refractory to Ko+ added after exposure to inside Ca2+. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside-out patches of red cell membranes, the open probability (Po) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37 degrees C. Net K+ efflux of intact red cells is also reduced by increasing temperature, as are the Po values of inside-out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important pathophysiologically, because it represents the major pathway for cell shrinkage via KCl and water loss that occurs in sickle cell disease.

Activation of Glut1 Glucose Transporter in Human Erythrocytes

Glut1, the only glucose transporter isoform expressed in the human red blood cell (RBC), binds to and is inhibited by cytochalasin B (CB). In the present study we show that incubation of RBC ghost membranes with 10 μM cytochalasin E (CE) results in a 1.8-fold increase in the number of glucose-sensitive cytochalasin B binding sites. Moreover, treatment with CE was associated with no observable change in the protein composition of RBC ghosts determined by SDS–PAGE. Removal of surface (“extrinsic”) proteins from RBC ghosts by treatment with 0.2 mM EDTA (pH 12) for 30 min resulted in a similar 1.8-fold increase in the number of glucose-sensitive CB binding sites. Western blot analysis showed that treatment with CE or EDTA resulted in no change in the amount or mobility of Glut1 present in ghost membranes. Glucose transport, measured as CB-inhibitable 3-O-[3H]methylglucose uptake by resealed ghosts, was stimulated in CE-treated resealed ghosts, with thet1/2to equilibrium decreasing from 6.8 ± 0.5 to 3.9 ± 0.3 s (P< 0.05). Treatment of ghosts with CE or EDTA followed by Western blotting of samples in the presence or absence of β-mercaptoethanol resulted in no change in immunoreactivity or mobility of the major Glut1 band. The above results suggest that a significant fraction of Glut1 transporters exists in an inactive form (“masked”) in RBC plasma membranes and that treatment of ghosts with CE or EDTA leads to an apparent activation of Glut1.

Imaging Analysis of Cellular Lipid Molecules Using Confocal Fluorescence Microscopy

Confocal fluocescence microscopy is regarded as a suitable tool for dynamic analysis of lipid molecules in living cells when it is combined with appropriate fluorescent lipid probes. At first, the advantates of confocal fluorescence microscopy are reviewed. Next, fluorescence labeling methods developed for biological studies, including fluorescent lipid probes, are surveyed. Then, following examples of imaging analysis of lipid dynamics with a confocal fluorescence microscope are introduced. 1) The biconcave morphology of red blood cell ghost membranes is observed as high resolution fluorescence images by using a water soluble fluorescent probe, rhodamine-dextran, and the fluorescent lipid probe, octadecyl rhodamine B. 2) Membrane fusion in single particles of the influenza virus with red blood cell ghost membranes is detected and quantitatively analysed through fluorescence dequenching of octadecyl rhodamine B. 3) Imaging analysis of lipid body formation in an oleaginous Mortierella fungus is undertaken using fluorescent analogues of phosphatidic acid and phosphatidylcholine.

Modification of Chloride Ion Transport in Human Erythrocyte Ghost Membranes by Photoaffinity Labeling Reagents Based on the Structure of 5-Nitro-2-(3-Phenylpropylamino)-benzoic Acid (NPPB)

Using human red blood cell ghost membranes, we have evaluated 5-nitro-2-[N-3-(4-azidophenyl)-propylamino]-benzoic acid and 5-nitro-2-[N-3-(4-azido-2,3,5,6-tetrafluorophenyl)-propylamino]-benzoic acid (FAzNPPB) as photoaffinity labeling agents based on the structure of the widely important Cl− channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB). The tetrafluoro-substituted aryl azide was found to be a more effective photoinactivating agent than the corresponding protio compound. Using a tritiated version ([3H]FAzNPPB), we demonstrated that photoinactivation of Cl− flux was accompanied by photolabeling of the band 3 protein and membrane lipids. Both processes were diminished in the presence of NPPB and the related arylanthranilate flufenamic acid. Photolabeling resulted in the incorporation of 1.0 ± 0.2 mol 3H/mol protein in the band 3 integral membrane domain, whereas the cytoplasmic domain was essentially unlabeled, By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, photolabeling was found to be the result of partial labeling of at least three different regions of the membrane domain, Based on trypsin proteolysis, reverse-phase high-performance liquid chromatography and electrospray ionization mass spectrometry analysis, it is proposed that one of the sites of photolabeling is the peptide lys-phe-lys (590-592), FAzNPPB is a successful polyfluoro aryl azide photoaffinity labeling agent which may be of further use in studying the diverse effects of arylanthranilates on biological membranes.

Loss of red blood cell glutathione during Mg deficiency: prevention by vitamin E, D-propranolol, and chloroquine

Mg deficiency results in loss of red blood cell glutathione and was thought to be due to decreased Mg-dependent synthesis. The effects of vitamin E, D-propranolol, and chloroquine on red blood cell glutathione levels in Mg-deficient rats were examined. Feeding the rats a Mg-deficient diet for 3 wk resulted in an approximately 80% decrease in serum Mg and a 55% loss of red blood cell glutathione; concomitantly, plasma thiobarbituric acid reactive (TBAR) materials rose 240%. All three drug treatments had no effect on the plasma Mg levels but significantly inhibited the rise in TBAR content and attenuated (60-80% effective) the loss of glutathione. Red blood cell ghost membranes from the Mg-deficient rats also exhibited 2.3-fold higher TBAR content, which was attenuated by vitamin E treatment. With isolated red blood cells from Mg-sufficient rats, loss of glutathione could be induced by a chemical oxyradical system. Direct protective effects were afforded by alpha-tocopherol and D-propranolol but not by chloroquine. The data suggest that 1) the loss of glutathione during Mg deficiency was due to increased oxidative degradation, 2) both vitamin E and D-propranolol protected by a membrane antiperoxidative action, and 3) chloroquine probably protected by diminishing prooxidant activity secondary to its inhibition of cytokine induction during Mg deficiency.

Red blood cell membrane dynamics in schizophrenia. II. Fatty acid composition

Fatty acid compositions were determined in red blood cell (RBC) ghost membranes of schizophrenic patients before and after haloperidol withdrawal, as well as with age-matched normal male control subjects ( n=22). Patients on haloperidol (HD) received treatment in doses between 5 and 20 mg/day for at least 5 weeks ( n=24). Drug-free patients ( n=19) were free of all psychotropic medications for an average of 40 days. A highly significant decrease in the levels (nmol/ml packed RBC) of polyunsaturated fatty acids (PUFAs), particularly 18:2( n−6) and 20:4( n−6), was found in both HD-treated and drug-free patients. This decrease remained in those patients who had withdrawn from HD for more than 5 weeks ( n=10). Concomitantly, the percentage of saturated and monoenoic fatty acids to total fatty acids increased significantly in schizophrenic patients. The resultant fatty acid profile consequently lowers the unsaturation index (UI), which represents the average number of double bonds per fatty acid molecule, in RBC ghost membranes of schizophrenic patients. Furthermore, the decreases in UI were significantly correlated to the increases in “structure order” of RBC ghost membranes as measured by the steady-state fluorescence anisotropy ( r s) in normal control and drug-free schizophrenic subjects. Therefore, it is likely that decreased levels of PUFAs in schizophrenic patients might result from a defective uptake of 18:2( n−6) into RBC membrane phospholipids. Since fatty acid composition of membrane phospholipids affects the relative degree of membrane fluidity, the present results lend further support that RBC membrane dynamics are altered in schizophrenia.

The effect of α-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membranelipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between α-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with α-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, α-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.

Erythrocyte membrane microviscosity and phospholipid composition in lead workers.

The microviscosity and fluidity of erythrocyte ghost membranes from lead workers and control subjects was measured by fluorescence polarisation using the fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). Increased lead was associated with a significant decrease in the average microviscosity of resealed and unsealed erythrocyte membranes. Since DPH fluorescence reflects the organisation of lipids in the central core of the membrane, two aspects of phospholipid metabolism were investigated. Phospholipids were extracted from red blood cell ghost membranes and identified by high performance liquid chromatography. The ratio of phosphatidyl choline to phosphatidyl ethanolamine, an established correlate of membrane fluidity, was significantly increased in lead workers. This is attributed to the known increases in red blood cell cholesterol in lead workers and the structural incompatibility of phosphatidyl ethanolamine and cholesterol, which result in a compensatory increase of phosphatidyl choline. Erythrocyte ghost membranes from control subjects were resealed with the intermediates in phospholipid synthesis that increase with a lead inhibited decrease in red blood cell pyrimidine 5'-nucleotidase. Membrane fluidity was not modified by incubation with cytidine triphosphate, uridine triphosphate, cytidine diphosphate choline, or cytidine diphosphate ethanolamine. Alterations in the microviscosity of the lipid regions of the hydrophobic core of the erythrocyte membrane bilayer and in the phospholipid composition of the membrane may be defects which contribute to the clinical and biochemical instability of the red blood cell on exposure to lead.

Kinetics of polymerization of a fluoresceinated derivative of complement protein C9 by the membrane-bound complex of complement proteins C5b-8.

The fluorescence self-quenching by energy transfer of FITC-C9, a fluoresceinated derivative of human complement protein C9 [Sims, P.J. (1984) Biochemistry (preceding paper in this issue)], has been used to monitor the kinetics of C9 polymerization induced by the membrane-associated complex of complement proteins C5b-8. Time-based measurements of the fluorescence change observed during incubation of FITC-C9 with C5b-8-treated sheep red blood cell ghost membranes at various temperatures revealed that C9 polymerization induced by the C5b-8 proteins exhibits a temperature dependence similar to that previously reported for the complement-mediated hemolysis of these cells, with an Arrhenius activation energy for FITC-C9 polymerization of 13.3 +/- 3.2 kcal mol-1 (mean +/- 2 SD). Similar measurements obtained with C5b-8-treated unilamellar vesicles composed of either egg yolk phosphatidylcholine (egg PC), dipalmitoylphosphatidylcholine (DPPC), or dimyristoylphosphatidylcholine (DMPC) revealed activation energies of between 20 and 25 kcal mol-1 for FITC-C9 polymerization by C5b-8 bound to these membranes. Temperature-dependent rates of C9 polymerization were observed to be largely unaffected by the phase state of membrane lipid in the target C5b-8 vesicles. The significance of these observations of the mechanism of C9 activation of membrane insertion is considered.

Evidence that spectrin binds to macromolecular complexes on the inner surface of the red cell membrane.

Spectrin binds to a population of high-affinity sites on the exposed surface of inverted vesicles prepared from human red blood cell ghost membranes. Optimal spectrin binding requires the presence of monovalent salts but does not require calcium or magnesium. The band 2 subunit of spectrin, prepared in SDS, can also bind to vesicles, but isolated band 1 is inactive. Pre-incubation of inverted vesicles with antibodies directed against the cytoplasmic segment of band 3 or against bands 4.1-4.2 inhibits the binding of spectrin to the same vesicles. Antibodies against the cytoplasmic portion of glycophorin A have no effect. These results suggest that spectrin binds to a protein acceptor on the cytoplasmic surface of the red cell membrane which is close to the cytoplasmic segments of bands 3 and 4.1 and/or 4.2.

Human erythrocyte anion exchange site characterised using a fluorescent probe.

A plasma membrane anion exchange system allows the red blood cell to replace intracellular bicarbonate rapidly with extracellular chloride to facilitate carbon dioxide release into the lungs. The protein responsible for this exchange is a glycoprotein of molecular weight about 93,000, called band 3, which comprises approximately 25% of the total red cell membrane protein and is present in the membrane as a non-covalent dimer1. The molecular mechanism of anion transport remains obscure, in spite of the characterisation of the extracellular anion binding site by a number of methods including interactions with disulphonic stilbene probes which act as specific inhibitors of anion exchange2. One of these compounds (4,4′-diisothiocyano-2,2′-disulphonic stilbene; DIDS) causes complete inhibition of anion exchange by reacting at approximately one site per band 3 monomer (∼2.5 nmol per mg protein)3. A fluorescent DIDS analogue, DBDS (4,4′-dibenzoamide-2,2′-disulphonic stilbene), binds reversibly and specifically to the DIDS reactive site where it also inhibits anion exchange6 . When bound, the fluorescence intensity of DBDS increases by two orders of magnitude and this has enabled us to investigate the binding kinetics of DBDS to red blood cell ghost membranes by fluorescence equilibrium and temperature-jump methods. Our equilibrium binding data indicate that there are two classes of DBDS binding sites on red cell ghosts. The reaction scheme that we present here suggests that the apparent high affinity (15–60 nM) for binding the first DBDS molecule is the result of a two step process: an initial binding of lower affinity (K1∼0.5 µM) to band 3 followed by a slow conformational change (∼5 s−1) in the binding site which acts to lock the DBDS molecule in place. A second DBDS molecule binds with low affinity (∼3 µM) to a site which is more difficult to characterise.

Freeze-fracture ultrastructural alterations induced by filipin, pimaricin, nystatin and amphotericin B in the plasma membranes of Epidermophyton, Saccharomyces and red blood cells. A proposal of models for polyene·ergosterol complex-induced membrane lesions

The effects of chemically different polyenes on fungal membranes Epidermophyton floccosum, a human pathogenic fungus, and Saccharomyces cerevisiae and human red blood cell membranes were studied by freeze-fracture electron microscopy in order to elucidate the interaction of these antibiotics with ergosterol. Each type of neutral, small amphoteric and large amphoteric polyenes produces a distinct morphological effect on the fungal membranes: 1. (1) Pit formation type. Filipin, a neutral polyene, produces 250–300 Å diameter “pits” or “invagination” both in ergosterol-containing fungal plasma membranes and cholesterol-containing red blood cell ghost membranes. 2. (2) Network particle aggregation type. The small amphoteric polyene, pimaricin, produces a network of membrane particle aggregation which encloses 1000 Å diameter particle-free areas in fungal membranes. These areas are slightly elevated toward the outside of the cell. 3. (3) Random particle aggregation type. The large amphoteric polyenes, amphotericin B and nystatin, cause a random segregation of the fungal plasma membrane and the red blood cell ghost membranes into particle-free and aggregated areas. It is concluded that these morphological differences are due to different mechanisms of polyene-sterol interactions in which the different size of the macrolide ring in the antibiotic structure may be involved. Since all of these antibiotics, except filipin, cause no alterations on whole red blood cells detectable by negative staining and freeze-fracture electron microscopy, it is possible that they have a higher affinity to ergosterol than cholesterol in membranes.