Structure of Leishmania donovani coronin coiled coil domain reveals an antiparallel 4 helix bundle with inherent asymmetry.

Coiled coils are ubiquitous structural motifs that serve as a platform for protein-protein interactions and play a central role in myriad physiological processes. Though the formation of a coiled coil requires only the presence of suitably spaced hydrophobic residues, sequence specificities have also been associated with specific oligomeric states. RhXXhE is one such sequence motif, associated with parallel trimers, found in coronins and other proteins. Coronin, present in all eukaryotes, is an actin-associated protein involved in regulating actin turnover. Most eukaryotic coronins possess the RhXXhE trimerization motif. However, a unique feature of parasitic kinetoplastid coronin is that the positions of R and E are swapped within their coiled coil domain, but were still expected to form trimers. To understand the role of swapped motif in oligomeric specificity, we determined the X-ray crystal structure of Leishmania donovani coronin coiled coil domain (LdCoroCC) at 2.2Å, which surprisingly, reveals an anti-parallel tetramer assembly. Small angle X-ray scattering studies and chemical crosslinking confirm the tetramer in solution and is consistent with the oligomerization observed in the full length protein. Structural analyses reveal that LdCoroCC possesses an inherent asymmetry, in that one of the helices of the bundle is axially shifted with respect to the other three. The analysis also identifies steric reasons that cause this asymmetry. The bundle adapts an extended a-d-e core packing, the e residue being polar (with an exception) which results in a thermostable bundle with polar and apolar interfaces, unlike the existing a-d-e core antiparallel homotetramers with apolar core. Functional implications of the anti-parallel association in kinetoplastids are discussed.

Cloning, overexpression, purification and crystallization of the CRN12 coiled-coil domain from Leishmania donovani.

Leishmania donovani coronin CRN12 is an actin-binding protein which consists of two domains: an N-terminal WD repeat domain and a C-terminal coiled-coil domain. The coiled-coil domain is 53 residues in length. Helix-helix interactions in general and coiled coils in particular are ubiquitous in the structure of proteins and play a significant role in the association among proteins, including supramolecular assemblies and transmembrane receptors that mediate cellular signalling, transport and actin dynamics. The L. donovani coronin CRN12 coiled-coil domain (5.8 kDa) was cloned, overexpressed, purified to homogeneity and the N-terminal 6×His tag was successfully removed by thrombin cleavage. Crystals of recombinant L. donovani coronin CRN12 coiled-coil domain were grown by vapour diffusion using a hanging-drop setup. Diffraction-quality crystals were obtained and data extending to 2.46 Šresolution were collected at 100 K on BM14, ESRF, Grenoble, France. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 118.0, b = 50.6, c = 46.0 Å, ß = 111.0°. Matthews coefficient (VM) calculations suggested the presence of 4-6 molecules in the asymmetric unit, corresponding to a solvent content of ∼33-55%, and are consistent with self-rotation function calculations.

Transbilayer phospholipid asymmetry in Plasmodium knowlesi-infected host cell membrane.

The membranes from normal and Plasmodium knowlesi-infected rhesus monkey erythrocytes (90 to 95 percent infected with early ring stage) were analyzed for transbilayer distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), by means of chemical and enzymatic probes. The external monolayer of the normal red cell membrane contained at least 68 to 72 percent of the total phosphatidylcholine and 15 to 20 percent of the total phosphatidylethanolamine. In the infected cell, the transmembrane phosphatidylcholine distribution appeared to be reversed, with only 20 to 30 percent of it being externally localized, whereas roughly equal amounts of phosphatidylethanolamine were present in the outer and inner surfaces. However, total phosphatidylethanolamine were present in the outer and inner surfaces. However, total phosphatidylserine in both the infected and normal red cells was exclusively internal. Unlike that in the normal intact cell, external phosphatidylethanolamine in the parasitized cell was readily accessible to phospholipase A2. These results indicate that significant changes in molecular architecture of the host cell membrane are the result of parasitization.

A hospital-based study on knowledge and attitude related to vitiligo among adults visiting a tertiary health facility of central India.

BACKGROUND: Vitiligo is one of the common stigmatizing dermatosis in the Indian society and the vitiligo patients have to face significant psychological hurt and social neglect. The severity of the stigma is related to the society's attitude and knowledge about it. AIMS AND OBJECTIVES: To document the prevalent knowledge and attitude in general public towards vitiligo patients, and to identify the determinants of good/poor knowledge and attitude. MATERIALS AND METHODS: A systematic random sampling technique was adopted to enroll 700 adult participants visiting an urban tertiary healthcare facility of central India. We developed a questionnaire to collect information on knowledge and attitude of the participants. A composite score was developed for good knowledge and attitude and performance of the participants was compared with the selected determinants. Data analysis was conducted by Stata software version 11. RESULTS: The overall knowledge score was good for 66.3% (95% confidence interval [CI]: 62.8%, 69.8%) of the participants. However, the score for attitude was comparatively poor i.e., only 16.9% (95% CI: 13.9%, 19.5%). None of the studied parameters could be significantly correlated with the knowledge score. Being married and being engaged in a health care related occupation were significant predictors of good attitude levels with P = 0.042 and 0.034 respectively, whereas female gender was the significant predictor for poor attitude with an odds ratio of 0.54 (95% CI: 0.33, 0.9) and P = 0.018. CONCLUSIONS: There were widespread myths prevalent about vitiligo in the studied population. The knowledge scores were better than attitude scores.

Carbamyl phosphatidylcholine--cholesterol interactions in unilamellar vesicles.

Small unilamellar vesicles formed from 1-palmitoyl-2-O-(N-(heptadec-8-cis-enyl)carbamyl)-sn-glycero-3-pho sphocholine (CMPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence of varying amounts of cholesterol have been studied using fluorescence polarization and NMR (1H and 13C) techniques. The fluorescence polarization and 1H-NMR data clearly indicate that the phospholipid packing order in CMPC bilayers is significantly greater than that in the POPC bilayers. The 13C-NMR chemical shift measurements show that this difference between the two phospholipids possibly arises due to the intramolecular hydrogen-bond formation between the -NH and the phosphate residues in the CMPC molecule. It is further shown that unlike POPC, the CMPC packing order is not much affected by including cholesterol in the phospholipid bilayers. These results demonstrate that introduction of one -NH residue adjacent to the C-2 carbonyl carbon in the POPC molecule could make its structure more ordered in the vesicles bilayer, and also would alter its interactions with cholesterol.

Transbilayer distributions of red cell membrane phospholipids in unilamellar vesicles.

The phospholipid organization in unilamellar vesicles comprised of various purified phospholipid components of monkey erythrocyte membrane was ascertained using phospholipase A2 and trinitrobenzenesulfonic acid as external membrane probes. The vesicles were formed by sonication or detergent dialysis and fractionated by centrifugation or gel permeation chromatography. Experiments were done to confirm that the phospholipase A2 treatments did not cause lysis or induce fusion of the vesicles. This enzyme hydrolysed only the glycerophospholipids in the outer surface of the vesicles. The amounts of the external phospholipids determined by this enzymatic method were verified using the chemical probe, trinitrobenzenesulfonic acid. The choline-containing phospholipids and phosphatidylethanolamine localized randomly in the two surfaces of sonicated vesicles (outer diameter, about 30 nm), whereas phosphatidylserine preferentially distributed in the inner monolayer. This phosphatidylserine asymmetry virtually disappeared in detergent dialysed vesicles (outer diameter, about 45 nm). Furthermore, inclusion of cholesterol in both the types of vesicles resulted in more random glycerophospholipid distributions across the plane of vesicles bilayer, presumably due to the cholesterol-induced increases in the size of vesicles. These results demonstrate that the transbilayer distribution of erythrocyte membrane phospholipids in unilamellar vesicles are controlled mainly by the surface curvature rather than by interlipid interactions, and therefore suggest that phospholipid-phospholipid and phospholipid-cholesterol interactions should not play any significant role in determining the membrane phospholipid asymmetry in red cells. It is proposed that this asymmetry primarily originates from differential bindings of phospholipids with membrane proteins in the two leaflets of the membrane bilayer.

Carbamyl analogs of phosphatidylcholines. Synthesis, interaction with phospholipases and permeability behavior of their liposomes.

A novel class of phospholipase-resisting phosphatidylcholine analogs, in which the C-2 ester group or both C-1 and C-2 ester groups have been replaced by carbomyloxy functions (Formula--see text), have been synthesized. These lipids were not degraded by phospholipase A2, while complete hydrolysis occurred with phospholipase C. Ultrasonic irradiation of the aqueous dispersions of the phospholipids in the presence as well as in the absence of cholesterol resulted in the formation of closed bilayer structures as evidenced by negative staining electron microscopy and also by their ability to entrap [14C]glucose. The leakage rates of glucose at 37 degrees C from liposomes of these compounds have also been measured. Liposomes consisting of 1,2-dipentadecanylcarbamyloxy-sn-glycero-3-phosphorylcholine were found to be more leaky (2.1%/h) as compared to the liposomes of 1-palmitoyl-2-pentadecanylcarbamyloxy-sn-glycero-3-phosphorylcholine (0.5%/h). Moreover, inclusion of cholesterol (33 mol%) into the bilayers of the former phospholipid had no effect on the leakage rate (2.4%/h) while it effectively reduced permeability of the latter (0.22%/h). These phosphatidylcholines are useful for studying the possible role of phospholipases in the capture and lysis of liposomes in vivo.

Out-to-in translocation of butanetriol-containing phospholipid analogs in human erythrocyte membrane.

Fluorescent butanetriol-containing phospholipid analogs were synthesized by replacing the glycerol moiety in 1-hexadecanoyl-2-[6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) aminohexanoyl]-sn-glycero-3-phosphocholine, -phosphoethanolamine, -phosphoserine and 1-hexadecanoyl-2-[12-N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)aminododecanoyl]-sn-glycero-3-phosphocholine, -phosphoethanolamine, -phosphoserine by the 1,3,4-butanetriol residue, and their out-to-in translocation in the human erythrocyte membrane studied by 'back exchanging' the outer surface-incorporated phospholipids using bovine serum albumin. The results of these studies indicate that the replacement of the glycerol moiety by the 1,3,4-butanetriol residue in aminophospholipids does not effect their out-to-in translocation in the human erythrocyte membrane. Furthermore, since earlier study by Arora and Gupta (Biochim. Biophys. Acta 1324 (1997) 47-60) has shown that the conformation of the 1,3,4-butanetriol phospholipids possess the backbone conformation similar to that of glycerophospholipids, it is suggested that besides the normal phospholipid polar head-group, a normal phospholipid interface conformation may also be required for the aminophospholipid-translocase interactions.

Glycerol backbone conformation in phosphatidylcholines is primarily determined by the intramolecular stacking of the vicinally arranged acyl chains.

To analyse the effect of the altered glycerol backbone structure on the glycerophospholipid conformation, we have replaced the glycerol moiety by the rac-1,2,4-butanetriol residue in 1,2-diacyl-sn-glycero-3-phosphocholines (PC), and then analysed the resulting 1,2-dialkanoyloxy-rac-but-4-yl-[2-(trimethylammonium)ethyl] phosphates (1,2-bPC) and 1,3-dial-kanoyloxy-rac-but-4-yl-[2-(trimethylammonium)ethyl] phosphates (1,3-bPC) by high-resolution 1H- and 13C-NMR spectroscopy in both CDCI3 and D2O. The preferred conformation about the C1-C2 glycerol bond in PC was almost completely preserved in 1,2-bPC, but it was completely random in case of 1,3-bPC. Out of the three C-C bonds present in the butanetriol backbone of 1,3-bPC, only the C2-C3 bond experienced a restricted rotation. However, the conformational preference about this bond was virtually similar to that observed for the C1-C2 bond in PC. These results clearly demonstrate that the preferred conformation of the glycerol backbone is determined primarily by the intramolecular acyl chain stacking which essentially requires a vicinal arrangement of the acyl chains in glycerophospholipids.

Novel thermal phase transition behavior of phosphatidylcholine analogs containing 1,2,4-butanetriol as their backbone.

The sn-glycerol moiety in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was replaced by the rac-1,2,4-butanetriol residue, and the aqueous dispersions of the resulting DPPC analogs, viz. 1,2-dihexadecanoyloxy-rac-but-4-yl-[2-(trimethyl-ammonium)ethyl]ph osphate (1,2-bPC) and 1,3-dihexadecanoyloxy-rac-but-4-yl-[2-(trimethylammonium)ethyl]pho sphate (1,3-bPC), were characterized by differential scanning calorimetry (DSC) and fluorescence polarization technique. Also, the corresponding (3R)-isomer of 1,3-bPC (1,3-(3R)bPC) was prepared and characterized by DSC. While the thermal phase transition properties of 1.2-bPC were similar to that of racemic DPPC, 1.3-bPC in identical conditions showed an abnormal property by exhibiting a metastable phase behavior at about 15 degrees C. This abnormal property was associated only with the racemic mixture and was completely absent in its 3R-isomer. 1,3-(3R)bPC, unlike DPPC, showed only two high enthalpy transitions at about 29.7 degrees C (delta H, 7.45 kcal/mol) and 35.3 degrees C (delta H, 6.93 kcal/mol). These results clearly demonstrate that an insertion of one additional methylene residue between the glycerol C1 and C2 carbons in DPPC markedly alters its thermal phase transitional properties, whereas these properties remain virtually unchanged if a similar chemical change is introduced between the glycerol C2 and C3 carbon atoms.

Influence of the phospholipid structure on the stability of liposomes in serum.

The effect of serum on the structural integrity of liposomes consisting of ether and/or carbamyl analogs of 1,2-diester phosphatidylcholine (PC) has been evaluated by measuring both the efflux of the entrapped 6-carboxyfluorescein and the lipid transfer to serum proteins, and the results have been compared with the egg PC liposomes. Replacement of the C-1 ester bond in PC by an ether linkage did not significantly enhance the liposome stability, but it was markedly increased upon introducing further structural changes in the C-2 ester region of the resulting 1-ether-2-ester PC. However, the stability was not influenced by altering the steric configuration of the latter phospholipid. These results strongly suggest that lysis of liposomes in serum can be prevented by structurally modifying the ester bond(s) in the phospholipid component of liposomes.

Effect of phospholipid structure on stability and survival times of liposomes in circulation.

The phosphatidylcholine (PC) component of liposomes was structurally modified by replacing its C-1, or both C-1 and C-2, ester linkage(s) with an ether and/or carbamyl bond(s) or by changing its steric configuration. Small unilamellar liposomes were formed from PC, traces of the corresponding 14C-labeled PC and cholesterol in the presence of 6-carboxyfluorescein (02.M) by sonication, and purified by centrifugation. These liposomes were administered intravenously to rats, and their stability in blood as well as the rate of their clearance from the circulation were determined. Stability and survival times of liposomes were markedly increased by modifying both the C-1 and the C-2 ester linkages in PC. A similar but quantitatively smaller effect was observed when only the C-1 ester linkage was modified. However, the stability remained unaffected by changing the steric configuration of PC, but this modification influenced the clearance rate of liposomes from the circulation. These results demonstrate that both stability in blood and the clearance rate from circulation can be modulated by structurally modifying the ester linkages in the phospholipid component of liposomes.

Metastable phase behavior of a sphingolipid analogue.

The phase behavior of the sphingolipid analogue 1-palmitoyl-2-tridecanylcarbamyloxy-sn-glycero-3-phosphocholine (CM-PC) has been studied by differential scanning calorimetry. When CM-PC is cooled at rates greater than 5 K/min, subsequent heating runs exhibit metastable behavior: a low enthalpy exotherm is observed at about 9 degrees C (delta H = -(1-2)kcal/mol), followed by a high enthalpy endotherm at 38 degrees C (delta H = 13 kcal/mol). Systematic variation of cooling/heating protocols indicates that CM-PC exhibits two low temperature states, one metastable and the other stable. Cooling from the liquid crystalline state results in formation of the metastable low-temperature polymorph I, which must transform into the stable low-temperature polymorph II before the liquid crystalline state can be reached again. This metastable thermal behavior is virtually identical to that recently reported for synthetic palmitoyl cerebroside (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966) and for bovine brain n-acylcerebrosides (Curatolo, W. (1982) Biochemistry, 21, 1761-1764). The observation of the metastable phase behavior of CM-PC indicates that the sphingosine backbone is not a prerequisite for such metastable behavior. Furthermore, the carbamyl group in CM-PC is reversed in orientation compared with the amide of sphingolipids (-NH-CO- vs. -CO-NH-), suggesting that the intermolecular hydrogen bonding potential, rather than some highly specific steric or conformational constraint, is responsible for the observed metastability of sphingolipids.

Modification of phospholipid structure results in greater stability if liposomes in serum.

Previous studies have revealed that the replacement of the C-2 ester group in phosphatidylcholine by the carbamyloxy function renders the resulting lipids, without affecting the properties of the liposomes, resistant to hydrolysis by phospholipase A2 (Gupta, C.M. Bali, A. (1981) Biochim. Biophys, Acta 663, 506-515). As an extension of this work, the effect of serum on the stability of liposomes, prepared from 1-palmitoyl-2-heptadec-10-cis-enylcarbamyloxyphosphatidylcholine (carbamylphosphatidylcholine), has been examined. The stability has been measured in terms of (a) bilayer permeability to solutes, and (b) the lipid transfer to serum proteins. Replacement of egg phosphatidylcholine in liposomes by the carbamyl analog prevented serum-induced leakage of the entrapped solutes and also inhibited the lipid (phospholipid and cholesterol) transfer. Manipulation of the cholesterol content of the liposomes had no effect on the stability. These observations indicate that the interaction of serum proteins with liposomes probably involves a highly specific binding of the proteins to the liposome surface.

Antibody-mediated targeting of liposomes to erythrocytes in whole blood.

F(ab')2 fragments derived from anti-rat erythrocyte antibody or normal rabbit serum IgG were covalently attached to the surface of liposomes consisting of equimolar amounts of egg phosphatidylcholine and cholesterol. These liposomes were interacted with rat, monkey or mouse blood, and their binding to both red and white blood cells was determined. Results of these studies show that coupling of liposomes to anti-rat erythrocyte F(ab')2 considerably enhances their binding to erythrocytes in rat blood. However, no such increase in the binding was observed with rat leukocytes or monkey and mouse erythrocytes. Besides, the interactions between the liposomes and target cells did not affect the permeability properties of the liposome bilayer. These observations indicate that liposomes coupled to cell-specific antibodies may serve as highly useful carriers for homing of drugs/enzymes to specific cells in biophase.

Membrane phospholipid organization in calcium-loaded human erythrocytes.

Intracellular Ca2+ levels in human erythrocytes were increased by incubating them with variable concentrations of Ca2+ in the presence of ionophore A23187. Experiments were done to confirm that the Ca2+ loading did induce changes in the cell shape and membrane protein composition. The effect of the increased cytoplasmic Ca2+ levels on the membrane phospholipid organization was analysed using bee venom and pancreatic phospholipases A2, Merocyanine 540 and fluorescamine as the external membrane probes. About 20% phosphatidylethanolamine (PE) and 0% phosphatidylserine (PS) were hydrolysed by the phospholipases in intact control cells, whereas in identical conditions these enzymes readily degraded, 20-30% PE and 7-30% PS, in Ca2+-loaded erythrocytes, depending on the cytoplasmic Ca2+ concentration. Also, Merocyanine 540 failed to stain the fresh or control erythrocytes, but it labeled the cells loaded with Ca2+. Furthermore, fluorescamine labeled approx. 20% PE in fresh or control erythrocytes while in identical conditions, significantly higher amounts of PE were modified in intact Ca2+-loaded cells. These results demonstrate that Ca2+ loading in human erythrocytes leads to loss of the transbilayer phospholipid asymmetry, and suggest that, together with spectrin, polypeptides 2.1 and 4.1 may also play an important role in maintaining the asymmetric distribution of various phospholipids across the erythrocyte membrane bilayer.

Possible basis for membrane changes in nonparasitized erythrocytes of malaria-infected animals.

Previous studies (Gupta et al. (1982) Nature 299, 259-261) have shown that nonparasitized erythrocytes of Plasmodium knowlesi-infected monkeys contain the procoagulant phospholipid phosphatidylserine (PS) in the outer-half of their membrane bilayer. A reinvestigation of this problem has now revealed that in acute P. knowlesi infection, at least 30% of the infected animals do not have this abnormality. However, PS externalization was a consistent feature in the uninfected red cells of chronically infected animals. Also, a similar membrane change was observed in the red cells of uninfected splenectomized monkeys. These results strongly suggest that spleen plays an important role in maintaining the exclusive inner distribution of PS in the normal erythrocyte membrane, and that partial migration of this lipid to the outer monolayer in nonparasitized erythrocytes could be attributed to an abnormal physiology of this organ in malarial infection.

Interaction of cholesterol with photoactivable phospholipids in sonicated vesicles.

Photoactivable phospholipids containing either alpha-diazo-beta-trifluoropropionyloxy or m-diazirinophenoxyl groups in the omega-positions of sn-2 fatty acyl chains were synthesized and incorporated into sonicated vesicles containing 33 mol% of cholesterol. Photolysis of the vesicles at 350 nm produced covalent cross-links between the synthetic phospholipids and cholesterol. The cross-linked products obtained using [14C]cholesterol were characterized by their chromatographic behavior, cleavage on phospholipase A2 treatment, base-catalyzed transesterification and mass spectral measurements. The cross-linking was shown not to involve the 3-beta-hydroxyl group of cholesterol, and it was concluded that the reactive carbene intermediates formed from the photolabels inserted into the hydrocarbon skeleton of cholesterol in the bilayer. The extent of cross-linking obtained was comparable to that observed previously using phospholipids alone, indicating that no lateral phase separation occurred. The present approach is promising for further precise studies of the molecular interactions between cholesterol and phospholipids in biological membranes.

Heat-induced alterations in monkey erythrocyte membrane phospholipid organization and skeletal protein structure and interactions.

Rhesus monkey erythrocytes were subjected to heating at 50 degrees C for 5-15 min, and the heat-induced effects on the membrane structure were ascertained by analysing the membrane phospholipid organization and membrane skeleton dynamics and interactions in the heated cells. Membrane skeleton dynamics and interactions were determined by measuring the Tris-induced dissociation of the Triton-insoluble membrane skeleton (Triton shells), the spectrin-actin extractability at low ionic strength, spectrin self-association and spectrin binding to normal monkey erythrocyte membrane inside-out vesicles (IOVs). The Tris-induced Triton shell dissociation and spectrin-actin extractability were markedly decreased by the erythrocyte heating. Also, the binding of the heated erythrocyte membrane spectrin-actin with the IOVs was much smaller than that observed with the normal erythrocyte spectrin-actin. Further, the spectrin structure was extensively modified in the heated cells, as compared to the normal erythrocytes. Transbilayer phospholipid organization was ascertained by employing bee venom and pancreatic phospholipases A2, fluorescamine, and Merocyanine 540 as the external membrane probes. The amounts of aminophospholipids hydrolysed by phospholipases A2 or labeled by fluorescamine in intact erythrocytes considerably increased after subjecting them to heating at 50 degrees C for 15 min. Also, the fluorescent dye Merocyanine 540 readily stained the 15-min-heated cells but not the fresh erythrocytes. Unlike these findings, the extent of aminophospholipid hydrolysis in 5-min-heated cells by phospholipases A2 depended on the incubation time. While no change in the membrane phospholipid organization could be detected in 10 min, prolonged incubations led to the increased aminophospholipid hydrolysis. Similarly, fluorescamine failed to detect any change in the transbilayer phospholipid distribution soon after the 5 min heating, but it labeled greater amounts of aminophospholipids in the 5-min-heated cells, as compared to normal cells, after incubating them for 4 h at 37 degrees C. These results have been discussed to analyse the role of membrane skeleton in maintaining the erythrocyte membrane phospholipid asymmetry. It has been concluded that both the ATP-dependent aminophospholipid pump and membrane bilayer-skeleton interactions are required to maintain the transbilayer phospholipid asymmetry in native erythrocyte membrane.

Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes.

Transbilayer phospholipid distribution, membrane skeleton dissociation/association, and spectrin structure have been analysed in human erythrocytes after subjecting them to heating at 50 degrees C for 15 min. The membrane skeleton dissociation/association was determined by measuring the Tris-induced dissociation of Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin extractability under low ionic conditions, and the binding of spectrin-actin with normal erythrocyte membrane inside-out vesicles (IOVs). The spectrin structure was ascertained by measuring the spectrin dimer-to-tetramer ratio as well as the spectrin tryptophan fluorescence. Both the Tris-induced Triton shell dissociation and the spectrin-actin extractability under low ionic conditions were considerably reduced by the heat treatment. Also, the binding of heated erythrocyte spectrin-actin to IOVs was significantly smaller than that observed with the normal cell spectrin-actin. Further, the quantity of spectrin dimers was appreciably increased in heat-treated erythrocytes as compared to the normal cells. This change in the spectrin dimer-to-tetramer ratio was accompanied by marked changes in the spectrin tryptophan fluorescence. In spite of these heat-induced alterations in structure and bilayer interactions of the membrane skeleton, the inside-outside glycerophospholipid distribution remained virtually unaffected in the heat-treated cells, as judged by employing bee venom and pancreatic phospholipase A2, fluorescamine and Merocyanine 540 as the external membrane probes. These results strongly indicate that membrane bilayer-skeleton interaction is not the major factor in determining the transbilayer phospholipid asymmetry in human erythrocyte membrane.