Antitrypanosomal activity of epi-polygodial from Drimys brasiliensis and its effects in cellular membrane models at the air-water interface

Epi-polygodial, a drimane sesquiterpene was isolated from Drimys brasiliensis (Winteraceae). This compound demonstrated high parasite selectivity towards Trypanosoma cruzi trypomastigotes (IC₅₀ = 5.01 μM) with a selectivity index higher than 40. These results were correlated with the effects observed when this compound was incorporated in cellular membrane models of protozoans, represented by Langmuir monolayers of dipalmitoylphosphoethanolamine (DPPE). Surface pressure-area isotherms showed that epi-polygodial expands DPPE monolayers at higher areas and condenses them at lower areas, which was attributed to the preferential interaction with the polar heads of the lipid. This mechanism of action could be corroborated with Polarization-Modulation Reflection-Absorption Spectroscopy and Brewster Angle Microscopy. These results pointed to the fact that the interaction of epi-polygodial with DPPE monolayers at the air-water interface affects the physical chemical properties of the mixed film, which may be important to comprehend the interaction of this drug with cellular membranes at the molecular level.     

Interaction of nominally soluble proteins with phospholipid monolayers at the air-water interface

The interactions of carbonmonoxyhemoglobin (HbCO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and polyhistidine with phospholipid monolayers at the air-water interface were studied at physiological pH and ionic strength. HbCO and GAPDH both interact more strongly with monolayers containing negatively charged lipids. The interaction of HbCO and GAPDH with lipid monolayers decreases with increasing pH. Both the HbCO-monolayer and the GAPDH-monolayer interactions can be modeled as diffusion-limited processes, with kinetic data fit to a stretched exponential equation. The significance of these kinetics are discussed. Polyhistidine interacts only with monolayers containing lipids with negatively charged headgroups. In total, the results presented are consistent with an HbCO-lipid interaction with a large electrostatic component, a GAPDH-lipid interaction with comparable electrostatic and hydrophobic components, and a polyhistidine-lipid interaction that is solely electrostatic.     

Peroxyl-oxidized erythrocyte membrane band 3 protein with anion transport capacity is degraded by membrane-bound proteinase

Human red blood cells anion exchange protein (band 3) exposed to peroxyl radicals produced by thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through band 3 was smaller indicating that removal achieved by proteinases includes oxidized band 3 which still retain transport ability. Proteinases recognize band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.     

Detection of the associated state of membrane proteins by polyacrylamide gradient gel electrophoresis with non-denaturing detergents Application to band 3 protein from erythrocyte membranes

Polyacrylamide gradient gel electrophoresis was carried out in micellar solutions of various detergents which differ in degree of potency to denature proteins. From the application of this method to band 3 protein from erythrocyte membranes, it was suggested that the procedure was useful in studying the molecular state of membrane proteins.The electrophoretic behaviors of human and bovine band 3 protein did not show any species specificity in either a denature state and a state resembling the native state. As well as in nonionic detergent solutions, the dimeric and tetrameric structures of bovine band 3 protein were preserved in sodium deoxycholate solution, in which protein complexes maintained in nonionic detergent solutions are frequently dissociated. Even in dodecyltrimethylammonium bromide solution, which is a denaturant for water-soluble proteins, part of the band 3 protein was still present as the oligomer. The results suggest that the oligomeric form of band 3 protein is the stable structure and that the dimer and tetramer possibly coexist in membranes.     

Oligomeric structure and the anion transport function of human erythrocyte band 3 protein

Conclusions Evidence from many laboratories using several different techniques strongly suggests that, in the intact red cell, band 3 exists as dimers which can associate with other dimers to form tetramers. The kinetics of anion transport inhibition by stilbenedisulfonates indicate that irreversible inhibition of one subunit does not detectably affect anion transport by the other subunit. This does not imply that monomeric band 3 could necessarily transport anions; the native conformation of each subunit may require stabilizing interactions with another subunit, as indicated by the recent work of Boodhoo and Reithmeier [10]. A more detailed understanding of the structure of the band 3 dimer/tetramer will require information on which specific segments of the primary structure are involved in subunit-subunit contact. The combination of chemical cross-linking with proteolysis [136] is a promising approach to this problem.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

The alteration of membrane proteins in human erythrocyte membranes induced by quinolinic acid,an endogenous neurotoxin Correlation of effect with structure

Quinolinic acid (2,3-pyridinedicarboxylic acid), an endogenous metabolite of l-tryptophan, reportedly via the kynurenine pathway, has been previously shown to possess neurotoxic properties when injected into rat striatum (Schwarcz R., Whetsell, W.O., Jr. and Mangano R.M. (1983) Science 219, 316–318) and to alter the physical state of human erythrocyte membrane proteins, as judged by ESR spectroscopy (Farmer, B.T., II and Butterfield, D.A. (1984) Life Sci. 35, 501–509). Both the morphologic and ESR studies employed nicotinic acid as one comparative control and found that the effect of quinolinic acid is significantly different from that of nicotinic acid. In the present study, we report that the effects of several structural analogues and positional isomers of quinolinic acid on the ESR parameter associated with the physical state of membrane proteins in human erythrocyte membranes suggest the following conclusions concerning the structure-effect relationship of quinolinic acid: The alteration in the conformation of membrane proteins: (1) requires the presence of two carboxylic acid groups; (2) is independent of their relationship to one another on the pyridine ring; (3) is slightly dependent on the presence of the pyridine nitrogen atom but is independent of the positional relationship of the two carboxylic acid moieties to the heteroatom; and (4) seems to depend upon the presence of restricted internal motion derived from the aromaticity in these compounds.     

Spin-label studies of the oligomeric structure of band 3 protein in erythrocyte membranes and in reconstituted systems

A spin-labeled fatty acid (16-doxylstearic acid), linked by an ester bond to a maleimide or a nitrene residue, was covalently attached to band 3 of erythrocyte membranes. The electron spin resonance spectrum of the spin-labeled protein was examined at different temperatures in: (a) whole erythrocyte ghosts; (b) ghosts depleted of spectrin and actin; (c) alkaline-treated ghosts; (d) vesicles made with purified band 3 reassociated with dimyristoylphosphatidylcholine. Most spectra are composite with a major component corresponding to a large overall splitting. The determination of the percentage of the immobilized component was carried out by pairwise subtraction. At low temperatures (1–7°C), the highest fraction of immobilized component was found in dimyristoylphosphatidylcholine vesicles (approx. 100%); alkaline-treated membranes had approx. 75% of the immobilized component at the same temperature; whole erythrocyte, spectrin/actin-depleted and spectrin/actin/ankyrin-depleted ghosts gave identical results (approx. 60% of immobilized component). The immobilized fraction decreased in all samples with increasing temperature or addition of a nonsolubilizing concentration of dodecyl octaethylene glycol monoether. In dimyristoylphosphatidylcholine vesicles, however, the modification in the ratio of the two components was obtained only above the lipid transition temperature (23°C). The strong immobilization of the spin-labeled lipid chain at all temperatures suggested trapping of the lipid chain between proteins. At low temperature, in dimyristoylphosphatidylcholine vesicles or in alkaline-treated ghosts, lipid-protein segregation is likely to take place. In whole erythrocyte ghosts, on the other hand, the large contribution of the motionally restricted component at physiological temperature indicates the oligomeric nature of band 3. Partial dissociation of the oligomers occurs as the temperature is increased, but the presence or absence of cytoskeletal proteins has no influence on the state of oligomerization of band 3.     

Protoporphyrin-induced photodynamic effects on band 3 protein of human erythrocyte membranes

In previous studies it has been shown that protoporphyrin-induced photodynamic effects on red blood cells are caused by photooxidation of amino acid residues in membrane proteins and by the subsequent covalent cross-linking of these proteins. Band 3, the anion transport protein of the red blood cell membrane, has a relatively low sensitivity to photodynamic cross-linking. This cannot be attributed to sterical factors inherent in the specific localization of band 3 in the membrane structure. Solubilized band 3, for instance, showed a similar low sensitivity to cross-linking. By extracellular chymotrypsin cleavage of band 3 into fragments of 60 000 and 35 000 daltons it could be shown that both fragments were about equally sensitive to photodynamic cross-linking. The 17 000 dalton transmembrane segment, on the other hand, was completely insensitive. Inhibition of band 3-mediated sulfate transport proceeded much faster than band 3 interpeptide cross-linking, presumably indicating that the inhibition of transport is caused by photooxidation of essential amino acid residues or intrapeptide cross-linking. A close parallel was observed between photodynamic inhibition of anion transport and decreased binding of 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H₂DIDS), suggesting that a photooxidation in the immediate vicinity of the H₂DIDS binding site may be responsible for transport inhibition.     

Membrane perturbations induced by the interactions of zinc ions with band 3 in human erythrocytes

Of group 12 metals, zinc is an essential element to maintain our life, but other metals such as cadmium and mercury are toxic in cellular activities. Interactions of these metals with biomembranes are important to understand their effects on our living cells. Here, we describe the membrane perturbations induced by these metals in human erythrocytes. Of these metals, Zn²⁺ ions only induced the erythrocyte agglutination. Histidine residues in extracellular domains of band 3 participated in Zn²⁺-induced agglutination. Interestingly, it was found that band 3-cytoskeleton interactions play an important role in Zn²⁺-induced agglutination. In contrast with Hg²⁺ and Cd²⁺ ions, Zn²⁺ ions greatly suppressed pressure-induced hemolysis by cell agglutination. Such a suppression was removed upon dissociation of agglutinated erythrocytes by washing, indicating the reversible interactions of Zn²⁺ ions with erythrocyte membranes. Excimer fluorescence of pyrene indicated that spectrin is denatured by a pressure of 200 MPa irrespective of hemolysis suppression. Taken together, these results suggest that the agglutination of erythrocytes due to the interactions of Zn²⁺ ions with band 3 is stable under pressure, but spectrin, cytoskeletal protein, is denatured by pressure     

The characterisation of two partially purified systems for Na+-dependent amino acid transport

Two systems mediating the transport of amino acids were studied in vesicles derived from protein-depleted membranes of pigeon erythrocytes. One system (ASC system) catalysed the Na⁺-dependent exchange of small neutral amino acids, such as alanine, serine and cysteine. The other system, also Na⁺-dependent, mediated the active transport of glycine. The ASC and glycine systems were distinguished by the sensitivity of the latter to the anion present, by the former's requirement for an exchangeable amino acid and by the inability of alanine to inhibit the transport of glycine. Preliminary results indicated that the influx of glycine was electrically silent. The only major integral protein retained in the vesicles was the band 3 protein, but that could not be unequivocally identified as the transporter.     

Rotational diffusion of band 3 proteins in membranes from En(a—) and neuraminidase-treated normal human erythrocytes

Recent experiments have demonstrated an association between band 3 and glycophorin A in the human erythrocyte membrane (Nigg, E.A., Bron, C., Girardet, M. and Cherry, R.J. (1980) Biochemistry 19, 1887–1893). Here, the influence of sialoglycoproteins on the rotational diffusion of band 3 in the human erythrocyte membrane was investigated by studying membranes from En(a—) and neuraminidase-treated erythrocytes. Rotational diffusion was measured by observing flash-induced transient dichroism of eosin-labeled band 3. Although erythrocytes of the rare phenotype En(a—) lack the major sialoglycoprotein, glycophorin A, no significant difference in band 3 rotation at pH 7.4 and 37°C could be detected between En(a—) and normal erythrocyte membranes. Band 3 rotation at pH 7.4 was also insensitive to the enzymatic removal of sialic acid from the surface of normal erythrocytes. Moreover, the existence of an essentially similar temperature-dependent equilibrium between band 3 proteins with different mobilities was observed in normal, En(a—) and neuraminidase-treated erythrocytes. From these results it is concluded that glycophorin A contributes less than 15% to the cross-sectional diameter of the band 3-glycophorin A complex in the plane of the normal membrane. The rotation of the complex at pH 7.4 is not significantly influenced by either steric or electrostatic interactions involving the oligosaccharide moiety of glycophorin A.     

Fine structure of the band 3 protein in human red cell membranes: Freeze-fracture studies

The major red cell membrane protein, band 3, is a glycoprotein which extends across the membrane from the extracellular space into the cytoplasmic compartment. It is widely held that band 3 is a component of the intramembrane particles (IMP) which can be demonstrated by freeze-fracture electron microscopy. In this study, we find that the outer surface poles of the IMP can be seen by freeze-etching after they are unmasked by proteolysis under conditions which excise the surrounding sialopeptides from the membrane. The poles appear as distinctive projections, 30–50 Å in diameter, the “ES particles.” The ES particles remain associated with the outer surface of the membrane following cleavage of the band 3 polypeptide by chymotrypsin or pronase. This is consistent with previous biochemical studies which have shown that the 38,000-dalton outer surface segment of band 3 is intercalated in the lipid bilayer. A granulofibrillar component at the inner surface of the membrane is provisonally identified as the 40,000-dalton inner-surface domain of band 3.     

Divalent cation enhancement of the agglutinability by soyabean lectin of liposomes prepared from total lipid of erythrocytes and of erythrocyte membranes

CaCl₂ or MgCl₂ but not NaCl enhances the soyabean lectin-induced agglutination of liposomes prepared from total lipids of erythrocyte membranes. The addition of purified phosphatidylserine to the total lipids of erythrocyte membranes before the formation of liposomes inhibits lectin-induced agglutinability of the preparation in the absence of CaCl₂, but not in its presence. When preformed phosphatidylserine liposomes are added to liposomes of total lipids of erythrocyte ghosts, they do not inhibit agglutination, indicating that phosphatidylserine does not inhibit the lectin directly. CaCl₂ or MgCl₂ but not NaCl also stimulates the soyabean lectin-induced agglutination of human erythrocyte membranes.Electron micrographs indicate that the liposome preparations are multilamellar and separate even in the presence of CaCl₂. When such liposomes are treated with lectin with or without CaCl₂, the electron micrographs show significant agglutination without apparent fusion. The reversal of the agglutination of liposomes by specific sugars followed by turbidimetric and electron microscopic techniques supports the conclusion that CaCl₂ stimulated lectin-induced agglutination is unaccompanied by fusion.The stimulation by divalent cations of lectin-induced agglutination of erythrocyte ghosts or of our liposomes may be due to a decrease in apparent surface charge of these membrane systems.     

Role of sulfhydryl groups in band 3 in the inhibition of phosphate transport across erythrocyte membrane in visceral leishmaniasis

Membrane destabilization in erythrocytes plays an important role in the premature hemolysis and development of anemia during visceral leishmaniasis (VL). Marked degradation of the anion channel protein band 3 is likely to allow modulation of anion flux across the red cell membrane in infected animals. The present study describes the effect of structural modification of band 3 on phosphate transport in VL using (31)P NMR. The result showed progressive decrease in the rate and extent of phosphate transport during the post-infection period. Interdependence between the intracellular ionic levels seems to be a determining factor in the regulation of anion transport across the erythrocyte membrane in control and infected conditions. Infection-induced alteration in band 3 made the active sites of transport more susceptible to binding with amino reactive agents. Inhibition of transport by oxidation of band 3 and subsequent reversal by reduction using dithiothreitol suggests the contribution of sulfhydryl group in the regulation of anion exchange across the membrane. Quantitation of sulfhydryl groups in the anion channel protein showed the inhibition to be closely related to the decrease of sulfhydryl groups in the infected hamsters. Downregulation of phosphate transport during leishmanial infection may be ascribed to the sulfhydryl modification of band 3 resulting in the impaired functioning of this protein under the diseased condition.     

The stereospecific d-glucose transport protein in cholate extracts of human erythrocyte membranes Molecular sieve chromatography and estimation of molecular weight

Cholate extracts of human erythrocyte membranes (Lundahl, P., Acevedo, F., Fröman, G. and Phutrakul, S. (1981) Biochim. Biophys. Acta 644, 101–107) were fractionated by molecular sieve chromatography on Sepharose 6B, and the size and molecular weight of the active d-glucose transporter were estimated. The eluent contained 10 or 12.5 mM cholate, since higher concentrations inactivated the glucose transporter, and lower concentrations resulted in aggregation. The chromatographic distribution of the transport activity was reproducible, but was broader than one would expect for a homogeneous component. In the presence of 20 mM EDTA and 5 mM dithioerythritol, a combination which affords a highly stable transport activity, a molecular weight of $\text{400 000 ± 20 000}$ (Stokes' radius 5.9 nm) was estimated for the smallest active component. This value represents an upper limit, since the molecular weight of a non-spherical component would have been overestimated, and since bound cholate was calculated to represent about 12% of the molecular weight. The activity was completely recovered upon rechromatography. In 10 mM EDTA and 10 mM 2-mercaptoethanol, the estimated molecular weight of the smallest active component was $\text{210 000 ± 15 000}$, and this component was not stable upon rechromatography in 10 mM EDTA and 10 mM 2-mercaptoethanol. In the absence of chelating and reducing agents, cholate extracts from membranes which had been kept for 5 days at 4°C showed three additional active components smaller than 200 000 in molecular weight. Most of the phospholipids eluted later than the active components of molecular weight 400 000 or 210 000, in all experiments. Electrophoretic analysis in dodecyl sulfate of the chromatographic eluents indicates that at least one of the band 3-polypeptides (nomenclature according to Steck, T.L. (1974) J. Cell Biol. 62, 1–19) is a component of the active transporter. This band 3-polypeptide, which we denote 3.3, has an apparent molecular weight of 88 000. The stable transporter of molecular weight 400 000 might be a tetramer of the 3.3-polypeptide. Alternatively, a dimer of the 3.3-polypeptide in complex with lipids might account for this molecular weight. If the 3.3-polypeptide is the transporter subunit and if it binds cytochalasin B with high affinity ($\text{1.8 · 10}{}^5$ sites/cell) the recovered activity per 3.3-polypeptide is around 40% A degradation product of the 3.3-component (possibly a 4.5-component) might account for the unstable active transporter of molecular weight 210 000.     

The transport of chloroquine across human erythrocyte membranes is mediated by a simple symmetric carrier

The kinetic properties of the mediated transport of chloroquine in human erythrocytes are investigated. The high rates of translocation across the cell membrane and high adsorbance properties to glass surfaces have led to the development of new techniques for measuring initial rates of transport. Three different methodological procedures are used to accomplish a complete kinetic characterization of the system. All measurements were done at 25°C. Under zero-trans conditions the system displays complete symmetry, the Michaelis constants being 39.2±2.4 μM for influx and 36.6±5.6 μM for efflux. The respective maximal velocities are 206.4±36.0 μM·min^?1 and 190.0±7.8 μM·min^?1. Under equilibrium-exchange conditions the Michaelis constant is 108.6±15.6 μM and the maximal velocity is 630.3±50.4 μM·min^?1. This 3-fold increase in both K and V over the zero-trans values indicates that the rate-limiting step in the transport of chloroquine is the movement of the unloaded carrier. The kinetic data are consistent with the prediction of a simple carrier model.     

Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport numbers (sulfate ions per band 3 per minute) served as a measure of functional recovery after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19–36) may to some extent reflect this lipid dependence.     

The solubilization of cytoskeletons of human erythrocyte membranes by p-mercuribenzene sulphonate

The disruption of erythyrocyte membrane cytoskeletons brought about by treatment with p-mercuribenzene sulphonate (PMBS) has been followed by measurements of turbidity and the binding of ²⁰³Hg-labelled PMBS. After pretreatment with N-ethylmaleimide to block readily reactive sulphydryl groups, incubation with [²⁰³Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal pK_a value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups.