Rigidifying Effect Research Articles

Molecular order and mobility within membranes made from fluorocarbon/fluorocarbon and fluorocarbon/hydrocarbon double-chain ether and amide phospholipids

The order of membranes formed by various ether- or amide-connected fluorocarbon/fluorocarbon or mixed fluorocarbon/hydrocarbon double-chain phosphocholines which derive, respectively, from glycerol or diaminopropanol or serine, has been investigated using fluorescence anisotropy of TMA-DPH, and compared to that of membranes made from fluorinated or conventional ester-connected phosphatidylcholines. In the gel phase, membrane order is almost not affected by the molecular structure of the phospholipid. By contrast, in the liquid-crystalline state, it decreases on going (i) from membranes made from the fluorocarbon/fluorocarbon phospholipids to those formed by mixed fluorocarbon/hydrocarbon phospholipids, then to conventional ones, (the rigidifying effects of the fluorinated tails, their number and length is thus becoming noticeable) or (ii) from membranes made from fluorinated diamidopropano- or diamidoserinophosphocholines to those made from ester- or ether-connected glycerophosphocholines. The ester- versus ether-connection or 1,2- versus 1,3-connection of the chains on glycerol have no detectable impact on membrane order.

Chiral sugar recognition by a diboronic-acid-appended binaphthyl derivative through rigidification effect

Abstract To obtain a “rigid” saccharide receptor with a high chiral discrimination ability two 2-boronylphenylmethyl groups were directly introduced into OH groups in (R)-2,2′-dihydroxy-1,1′-binaphthyl. This compound (R)- 5 increased its fluorescence intensity upon the formation of intramolecular 1:1 complexes with saccharides, mainly because of the “rigidification effect”. The association constants (K) could be determined from plots of the fluorescence increase against the saccharide concentration. It was found that when the K difference between enantiomers is relatively small ( K D K L = 1.1 1.0 ∼ 1.0 1.7 ), the enantiomer with the larger fluorescence increase shows the smaller K. On the other hand, xylose and talose, which resulted in a chiral discrimination ability of 8.7-fold and 2.0-fold, respectively, showed the reverse fluorescence-K relationship. It is noteworthy that the chiral discrimination factor for xylose is the highest among those achieved so far.

Membrane properties and lipid peroxidation in food restricted animals.

Food restriction (FR) is a well-recognized method of extending mean and maximum longevity of rodents, but the mode of its action remains to be uncovered. This article reviews the effect of FR on the physical-chemical properties and lipid peroxidizability of cellular membranes. FR prevents the age-dependent increase in microviscosity and peroxidizability of cellular membranes. It has been suggested that a decrease in the body temperature occurring in undernourished animals may play a fundamental role in the process. Indeed, the lowering of average body temperature occurring in FR animals may induce a modification in membrane lipid composition, stimulating the cells to counteract the rigidifying effect of lower temperature. Thus, membranes are maintained in a proper functional state by a mechanism similar to that found in poikilotherm animals.

The effect of cholesterol in small amounts on lipid-bilayer softness in the region of the main phase transition

The temperature dependence of the small-angle neutron scattering from aqueous multilammellar DMPC lipid bilayers, containing small amounts of cholesterol, is analyzed near the main phase transition by means of a simple geometric model which yields the lamellar repeat distance, the hydrophobic thickness of the bilayer, the interlamellar aqueous spacing, as well as fluctuation parameters. The observation of anomalous swelling behavior in the transition region is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. Our results indicate that the effect of small amounts of cholesterol, ≲3 mole%, is a softening of the bilayers in the transition region, whereas cholesterol contents above this range lead to the well-known effect of rigidification. The possible biological relevance of this result is discussed.

Interaction of sodium alkyl sulphates with egg yolk lecithin vesicles / Wechselwirkungen von Na-Alkylsulfaten mit Vesikeln aus Eigelblecithin

The interaction of three anionic surfactants - sodium alkyl (decyl-, dodecyl-, tetradecyl-) sulphates - with unilamellar liposomes from egg yolk lecithin was studied fluorometrically by monitoring the leakage of the marker 6-carboxyfluorescein. An increase in leakage rate was observed with increasing surfactant concentration ; very small quantities of surfactant depending on its alkyl chain could disrupt the vesicles thus release the whole entrapped marker immediately after mixing with vesicles. Light scattering results suggest an increase in vesicle size followed by the formation of mixed micelles of surfactant and phospholipid molecules. The presence of cholesterol in the vesicle decreased the leakage of the marker due to its rigidifying effect.

Zeaxanthin (dihydroxy-β-carotene) but not β-carotene rigidifies lipid membranes: a 1H-NMR study of carotenoid-egg phosphatidylcholine liposomes

1H-NMR technique was applied to study liposomes formed with egg-yolk phosphatidylcholine containing as an additional component two carotenoid pigments: β-carotene or zeaxanthin (dihydroxy-β-carotene). A strong rigidifying effect of zeaxanthin but not of β-carotene with respect to hydrophobic core of lipid bilayer was concluded from the carotenoid-dependent broadening of the NMR lines assigned to -CH 2- groups and terminal -CH 3 groups of lipid alkyl chains. A similar effect of zeaxanthin with respect to polar headgroups was concluded on the basis of the effect of the pigment on the shape of NMR lines attributed to -N +(CH 3) 3 groups. In contrast, β-carotene increases motional freedom of lipid polar headgroups. The inclusion of both carotenoids to liposomes resulted in the enhanced penetration of Pr 3+ ions to the polar zone of the external layer of a membrane monitored by the splitting of the -N +(CH 3) 3 signal, the effect of β-carotene being much more pronounced. Differences in the effect on membrane structure and molecular dynamics observed for β-carotene and its polar derivative are discussed in terms of organization of a carotenoid-containing lipid membrane.

Effects of Fumonisin B1and (Hydrolyzed) Fumonisin Backbone AP1on Membranes: A Spin-Label Study

Electron spin resonance (ESR) spectroscopy and spin label techniques have been used to study the effects of fumonisin B1(FB1) and hydrolyzed fumonisin backbone (AP1) on the structural and dynamic properties of phosphatidylcholine membranes at the molecular level. Multilamellar liposomes consisting of dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (EYPC) were used. Six different nitroxide spin labels were used to determine what effects FB1may impart on the ordering and mobility of lipids in membranes. The experimental results disclose the following: (1) In the fluid phase membrane, FB1significantly increases the fluidities ofn-doxylstearic acid (SA) spin labels (SL) attached to carbons 5 and 7, which disorders the alkyl chains and perturbs the surface region of the bilayer; by comparison, minimal effects were detected near the center of the bilayer. (2) In the gel phase, FB1and AP1imparts marked rigidifying effects on membrane fluidity, which enlarges the change in ordering on the phase transition even further. (3) FB1also restricts the mobility of the (rigid) cholestane spin label. (4) A reduction in mobility of the tempo-stearate spin label suggests that the tricarballylic acid (TCA) moieties of FB1might mimic the structure of polar headgroups in phospholipids. The present results may provide additional mechanisms to elucidate the toxicological activities of the fumonisins.

Solutes in small amounts provide for lipid-bilayer softness: cholesterol, short-chain lipids, and bola lipids

The effect of the incorporation of small amounts (∼1 mole%) of amphiphilic solutes, such as cholesterol, a short-chain lipid (DC10PC), and a bola lipid, into multilamellar DMPC bilayers is studied by small-angle neutron scattering and differential-scanning calorimetry. The anomalous swelling behavior observed in the transition region of pure DMPC bilayers is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. Small amounts of the solutes are found to maintain or even enhance the bilayer softness. In the case of cholesterol, a systematic study shows that the well-known rigidification effect is observed only for cholesterol concentrations above 3–4 mole%. The results are discussed in relation to the physical properties of internal cell membranes.

Solid-Phase Synthesis of the Exposed Capsid Peptide VP2(96-107) of the Hepatitis A Virus. Study of Its Physicochemical Interactions with Phospholipids

The synthesis on a solid support of a dodecapeptide containing a HAV-VP2 exposed sequence is described. The surface activity of this peptide as well as its miscibility and interactions with dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), and phosphatidylinositol (PI) were determined. Interaction energies calculated applying the Gibbs equation gave very small values, thus suggesting either ideal miscibility or weak interactions. In all cases the electrical charge in DPPE and PI monolayers seems to create a high electrostatic potential, decreasing its interaction with any external molecule. This peptide was able to accommodate a certain amount of anilinonaphthalenesulfonate molecules, showing an intermediate hydrophobic character. Moreover, after incubation with saturated liposomes, the peptide showed a clear rigidifying effect at the level of polar heads, especially at temperatures under the transition temperature.

Molecular order and mobility within liposomal membrane made from highly fluorinated phospholipids

The temperature-dependent lipid structure, order and dynamics of membranes formed by fluorinated DF nC mPC phosphatidylcholines, as compared to their classical DMPC, DPPC and DSPC analogs, have been investigated using fluorescence anisotropy of an extrinsic fluorescent probe, i.e. 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). At low temperatures and in the gel phase of the liposomal membranes, membrane fluidity was almost unaffected by the fluorocarbon or hydrocarbon nature of the bilayer. In contrast, in the liquid-crystalline state, fluidity decreased substantially near the membrane's surface when fluorinated chains were present. The most important rigidifying effect was observed for a chain ended by a C 8F 17 tail as in DF 8C 5PC. It was also found that an acyl chain ended by a fluorinated tail as short as C 4F 9 e.g. inDF 4C 11PC, induced, in the fluid state a rigidifying effect similar to that of cholesterol.

Perturbation of Red Blood Cell Membrane Rigidity by Extracellular Ligands

It is known that binding of extracellular antibodies against the major sialoglycoprotein, glycophorin A, reduced the deformability of the red blood cell membrane. This has been taken to result from new or altered interactions between the glycophorin A and the membrane skeleton. We have shown by means of the micropipette aspiration technique that antibodies against the preponderant transmembrane protein, band 3, induce similar effects. A definite but much smaller reduction in elasticity of the membrane is engendered by univalent Fab fragments of the anti-band 3 antibodies. By examining cells genetically devoid of glycophorin A or containing a variant of this constituent, truncated at the inner membrane surface, we have shown that the anti-band 3 antibodies do not act through the band 3-associated glycophorin A. We examined the effect of anti-glycophorin A antibodies on homozygous Wr(a+b-) cells, in which an amino acid replacement in band 3 annihilates the Wright b (Wrb) epitope (comprising sequence elements of glycophorin A and band 3) and thus, by implication disrupts or perturbs the band 3-glycophorin A interaction; these cells show a much smaller response to an anti-glycophorin A antibody than do normal controls. We infer that in this case anti-glycophorin A antibodies exert their rigidifying effect through the associated band 3. Another anti-glycophorin A antibody, directed against an epitope remote from the membrane surface, however, increases the rigidity of both Wr(a+b-) and normal cells. This implies that not all antibodies act in the same manner in modifying the membrane mechanical properties. The effect exerted by anti-band 3 antibodies appears not to be transmitted through the band 3-ankyrin-spectrin pathway because the rigidifying effect of the intact antibody persists at alkaline pH, at which there is evidence that the ankyrin-band 3 link is largely dissociated. The large difference between the effects of saturating concentrations of the divalent and univalent anti-band 3 antibodies implies the existence of an overriding effect on rigidity, resulting from the bifunctionality of the intact antigen. Freeze-fracture electron microscopy shows that the anti-band 3 promotes the formation of small clusters of intra-membrane proteins. Extracellular ligands may in general act by promoting strong or transient interactions between integral membrane proteins, thereby impeding local distortion of the membrane skeletal network in response to shear.

Heterogeneity of microsomal membrane fluidity: evaluation using intrinsic tryptophan energy transfer to pyrene probes

Membrane fluidity measurements based on excimer formation of pyrene and pyrene derivatives as a measure of lateral diffusion yield a decreased fluidity in the presence of proteins [1–3]. It was the aim of our study to investigate whether the reduced excimer formation is due to a rigidifying effect of proteins on the whole membrane or if the fluorophore mobility is mainly hindered in the immediate protein environment. Resonance energy transfer in microsomal membranes between intrinsic tryptophan residues and pyrene were used to study the excimer formation rate in the vicinity of proteins. The excimer-to-monomer fluorescence ratio at excitation via resonance energy transfer is lower than that observed for the direct excitation. The results suggest that, because of a reduced fluidity in the neighbourhood of proteins, pyrene and pyrene fatty acids do not diffuse homogeneously in the membrane plane. A fluidity gradient exists from the membrane proteins to the bulk lipid.

Synthesis and aggregative properties of GM1 ganglioside (IV 3Neu5AcGgOse 4Cer) containing d-(+)-2-hydroxystearic acid

GM1 ganglioside containing a hydroxylated fatty acid moiety, GM1(OH), was synthesized starting from lyso-GM1 and d-(+)-2-hydroxystearic acid. The aggregative, geometrical and distribution properties of GM1(OH) were compared with those of stearic acid containing GM1 ganglioside; laser light scattering measurements, differential scanning calorimetry and fluorescence spectroscopy were used. GM1 and GM1(OH) are present in solution as micelles with a hydrodynamic radius of 58.7 and 60.0 Å, and molecular mass of 470 and 570 kDa, respectively. The surface area occupied by the monomer of GM1(OH) at the lipid-water interface of the aggregate was calculated to be 117 Å 2, which is 3 Å 2 lower than that determined for GM1. Proton NMR analyses of GM1 and GM1(OH) suggest different three-dimensional structures at the ganglioside lipid-water interface. Both GM1(OH) and GM1 inserted into dipalmitoylphosphatidylcholine (DPPC) vesicles undergo segregation phenomena, with the formation of ganglioside-enriched microdomains, but GM1(OH) shows a higher degree of dispersion in the DPPC matrix and exerts a lower rigidifying effect than does GM1.

Hydrophobic mismatch and long-range protein/lipid interactions in bacteriorhodopsin/phosphatidylcholine vesicles.

Mismatch between the hydrophobic thicknesses of transmembrane proteins and the supporting lipid bilayer and its consequences on the lateral organization of lipids have been investigated with bacteriorhodopsin and phosphatidylcholine species with a variety of acyl-chain lengths. The purple membrane, from the bacterium Halobacterium halobium, was used and reconstituted with dilauroyl-(Lau2GroPCho), dimyristoyl- (Myr2GroPCho), dipalmitoyl- (Pam2GroPCho) and distearoyl- (Ste2GroPCho) glycerophosphocholine. The phase behaviour of the lipids was investigated at different temperatures and different protein/lipid molar ratios, by analyzing the fluorescence excitation spectra of the 1-acyl-2-[8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine probe, and by measuring the fluorescence depolarization of the 1,6-diphenyl-1,3,5-hexatriene probe. Data obtained with 1-acyl-2-[8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine shows that bacteriorhodopsin produced positive or negative shifts in the phase transition temperature of the host lipids depending on the strength and sign of the mismatch between the lipid and protein hydrophobic thicknesses and also on the protein concentration and aggregation state in the lipid bilayer. In the region of high protein concentration (bacteriorhodopsin/phosphatidylcholine molar ratios approximately 1:50) and despite the presence of the endogenous lipids, bacteriorhodopsin (hydrophobic length dP approximately 3.0-3.1 nm) brought about a large upward shift in the phase-transition temperature of Lau2GroPCho (delta T approximately 40 K, mean hydrophobic thickness d approximately 2.4 nm), and to a lesser extent of Myr2GroPCho (delta T approximately 23 K, d approximately 2.8 nm), accounting for a strong rigidifying effect of the protein on these short-chain lipids. Bacteriorhodopsin had no influence on the phase properties of Pam2GroPCho (delta T approximately 0 K, d approximately 3.2 nm), a lipid whose mean hydrophobic thickness is similar to that of the protein. In contrast, the transition temperature of Ste2GroPCho was decreased (delta T approximately -13 K, d approximately 3.7 nm), indicating a fluidifying effect of the protein on this long-chain lipid. Similar effects on the lipid acyl-chain order were observed in the region of high-protein dilution (bacteriorhodopsin/phosphatidylcholine molar ratios < 1:500). In this region and for Lau2GroPCho, both the spectroscopic data and circular-dichroism spectra indicated that the protein was in the monomeric form. Phase diagrams, in temperature versus bacteriorhodopsin concentration, were constructed for Lau2GroPCho and Ste2GroPCho. On account of microscopic theoretical models and of the relative values of dP and d, these diagrams indicate a preference of the protein for those lipid molecules which are in the gel-ordered state in Lau2GroPCho but in the liquid disordered state in Ste2GroPCho.(ABSTRACT TRUNCATED AT 400 WORDS)

Pyrene excitation via resonance energy transfer from protein tryptophan reveals a fluidity gradient in liver microsomes

Membrane fluidity measurements based on excimer formation of pyrene and pyrene derivatives as a measure of lateral diffusion yield a decreased fluidity in the presence of proteins [1,2]. It was the aim of our study to investigate whether the reduced excimer formation is due to a rigidifying effect of proteins on the whole membrane or if the fluorophore mobility is hindered mainly in the immediate protein environment. Resonance energy transfer in microsomal membranes between intrinsic tryptophan residues and pyrene was used to study the excimer formation rate in the vicinity of proteins. The excimer-to-monomer fluorescence ratio after excitation via resonance energy transfer decreased compared to that observed for the direct excitation. The results suggest that, because of the reduced fluidity in the neighborhood of proteins, pyrene and pyrenedodecanoic acid do not diffuse homogeneously in the membrane plane.

A fluorescence anisotropy study of stabilizing effect of tri- and tetra- nitrovasodilatory drugs on DPPC liposomal membrane

Glyceryl trinitrate (GT) and pentaerythritol tetranitrate (PT) are two vasodilatory drugs. The physical properties of the membrane lipid matrix, which determine the structure and function of the membrane-bound proteins, generally control the pertubation mechanism of these drugs. Thus, physical interaction of these drugs with membrane lipids is very crucial for their clinical use, different cellular processes, as well as for targetted drug delivery systems. In the present paper, we have reported for the first time the interaction between these drugs and the lipid molecules in the liposomal system of dipalmitoylphosphatidylcholine (DPPC), as measured by steady-state fluorescence anisotropy using 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probe. Our results show that by dissolving in the lipid matrix these two drugs effectively stabilise the liposomal membrane: the effect being more in case of GT than in PT, indicating that the rigidifying effect is independent of the number of nitrate groups of the two drugs. This effect increases with the increase in drug concentration, implying solubilisation of all drug molecules. Though our in vitro study has more physical significance than a physiological one, the results obtained here may be used to interpret the effects that are observed in vivo.

Effect of ethanol on rat gastric surfactant: A fluorescence polarization study

Background: Gastric surfactant is believed to protect gastric mucosa by the hydrophobic properties of its phospholipidic component, which are reflected in the fluorescence polarization of a lipophylic fluorescent probe. The present study aimed to observe the consequences of intragastric administration of 40% ethanol on the physical properties of rat gastric surfactant. Methods: Fluorescence polarization studies and lipid composition of gastric mucosal surface scrapings were performed. Results: Time course experiments indicated that the ulcerogenic action of ethanol occurred along with a fluidization of the surface scrapings followed by secondary rigidification. The fluidizing effect of ethanol was related to modifications of the molecular dynamics of lipid structures. The rigidifying effect of ethanol was a result of an increase in the cholesteroltriglyceride and cholesterol-phospholipid ratios and an increase in the percent composition of phosphatidylethanolamine of surface scrapings. Conclusions: The results suggest that alcohol could alter the gastric mucosal barrier by its disorganizing effect on the molecular dynamics of the gastric surfactant. The second rigidifying effect of ethanol could be a part of the damage repair phenomenon.

Molecular motions and thermotropic phase behavior of triacylglycerols and cholesterol esters in herpesvirus-infected arterial smooth muscle cells: A deuterium nuclear magnetic resonance study

The physical state of lipids in arterial smooth muscle cells (SMC) may contribute to lipid accumulation following injury. We have previously demonstrated that herpes simplex virus (HSV) infection alters the physical state of the neutral lipid accumulating in arterial SMC, as determined by differential scanning calorimetry (Biochem. J. 268 (1990) 693–697). To more precisely determine the molecular packing of neutral lipids in HSV-infected cells, the influence of HSV-infection on the thermotropic and phase-behavior of the lipids in intact arterial smooth muscle cells and in cell-free lipid extracts was evaluated using [ 2H]-NMR, employing U-[ 2H]-oleic acid incorporated into cells. Inspection of the [ 2H]-line-widths indicate that the lipid of HSV-infected cells exhibited more restricted motion or a greater chemical shift dispersity than lipids from uninfected cells, as evidenced by significant broadening of the CDCD signals at 25°C and 45C. Fatty acid compositional analysis of the neutral lipids of control and HSV-infected cells following C18:1 supplementation (an amount added similar to the NMR experiments) reveals that: (1) there is approximately 55-fold more triacylglycerols (TG) than cholesteryl esters (CE) in control cells and 40-fold more TG than CE in the HSV-infected cells; (2) HSV infection significantly increases the C18:1 content of CE, and C18:3 and C20:4 in TG; and (3) HSV-infection does not alter the ratio of TG to CE. These data support the hypothesis that the greater restriction of the neutral lipids in HSV-infected cells may be due to the rigidifying effects of C18:1 on lipid mobility. Thus, alterations in the physical state of neutral lipids in HSV-infected cells may lead to reduced CE hydrolysis which, in turn, may contribute to or exacerbate lipid accumulation.

Thermotropic behavior of fatty acid ethyl esters in phospholipid liposomes

The thermotropic behavior of a series of synthetic fatty acyl ethylesters (FAEE) in multilamellar liposomes has been studied by differential scanning calorimetry and monitoring the changes in polarization emitted by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Their thermotropic behaviour has been compared to that of the homologous fatty acids (FA) from which they are synthesized in vivo in the presence of ethanol. Compared to the correspondent FA, saturated FAEE show, depending on the chain length, a minor rigidifying effect or even a slight fluidizing effect on phospholipid bilayers. Unsaturated FAEE show, compared to the homologous FA, slightly greater fluidizing properties. The difference between FA and FAEE is more evident in single component phospholipid liposomes in the gel phase, and in mixed liposomes of two lipids at temperatures at which microdomains of gel and liquid zones coexist. The calorimetric data suggest that FAEE are completely miscible with phospholipids both in the gel and liquid phases; they appear to destabilize the bilayer wherein the ethoxy head group interferes with the intrinsic phospholipid interactions.

Molecular order and fluidity of the plasma membrane of human platelets from time-resolved fluorescence depolarization

The ability of seven fluorescence polarization probes (1,6-diphenyl-1,3,5-hexatriene, 1-[(4-trimethyl- amino)phenyl]-6-phenyl-1,3,5-hexatriene, (2-carboxy- ethyl)-1,6-diphenyl-1,3,5-hexatriene, 16(9-anthroyloxy)-palmitic acid, CIS-parinaric acid, trans-parinaric acid and perylene) to report changes induced by temperature and Ca2+ in the plasma membrane of human platelets has been examined. The steady-state fluorescence anisotropy of the probes was compared after being incorporated into whole resting platelets, fragments of platelet plasma membrane and multilayers of lipids extracted from these membranes. In addition, we have investigated the molecular order and dynamics of the three preparations by time-resolved fluorescence depolarization of DPH and CE-DPH as a function of temperature and Ca2+ concentration. The high values of the order parameters found in intact platelets (SDPH, 36 degrees C = 0.70) were almost identical to those in membrane fragments and lipid vesicles, suggesting that lipid-lipid interactions and, therefore, the lipid composition are the main factors influencing the probe order parameter. Other lipid interactions such as those with membrane proteins and intracellular components have little effect on the SDPH in platelets. These measurements also showed that the stationary fluorescence anisotropy of DPH and CE-DPH in platelets is largely determined (80%) by the structural order of the lipid bilayer. Therefore, the previous "microviscosity" values based on stationary anisotropy data reflect the alignment and packing rather than the mobility of the bilayer components. The dynamic component of the anisotropy decay of these probes was analyzed in terms of the wobbling-in-cone model, allowing an estimation of the apparent viscosity of platelet plasma membrane (eta DPH, 36 degrees C = 0.5 P) that is similar to that of the erythrocyte membrane. This value decreased substantially in multilayers of native lipids, indicating a large effect of the lipid-protein interactions on the probe dynamics within the bilayer. When the temperature was raised from 25 degrees to 36 degrees C a pronounced decrease was observed in the order parameter and apparent viscosity, followed by a tendency to level-off in the 36 degrees-40 degrees C interval. This may be related to the end-point of the lipid phase separation reported by Gordon et al. (1983). Finally, the rigidifying (lipid ordering) effect of Ca2+ on the platelet plasma membrane could also be observed by the fluorescence anisotropy measurements, in the form of an increase (approximately 2%) of the order parameter of CE-DPH for Ca2+ concentrations in the millimolar range.