Effects of cholesterol on lipid organization in human erythrocyte membrane

The molar ratio of cholesterol to phospholipid (C/P) in human erythrocyte membrane is modified by incubating the cells with liposomes of various C/P ratios. The observed increase in cell surface area may be accounted for by the addition of cholesterol molecules. Fusion between liposomes and cells or attachment of liposomes to cells is not a significant factor in the alteration of C/P ratio. Onset temperatures for lipid phase separation in modified membranes are measured by electron diffraction. The onset temperature increases with decreasing C/P ration from 2 degrees C at C/P = 0.95 to 20 degrees C at C/P = 0.5. Redistribution of intramembrane particles is observed in membranes freeze-quenched from temperatures below the onset temperature. The heterogeneous distribution of intramembrane particles below the onset temperature suggests phase separation of lipid, with concomitant segregation of intramembrane protein into domains, even in the presence of an intact spectrin network.     

Imipramine and lipid phase transition in inner mitochondrial membrane

As ascertained by freeze-fracture electron microscopy, imipramine prevents lateral phase separation from taking place in inner mitochondrial membranes at sub-zero temperatures. Electron spin resonance (ESR) measurements performed on mitochondrial membranes labeled with the N-oxyl-4′,4′-dimethyloxazolidine derivative of 16-ketostearic acid, show that the spin probe motion is markedly inhibited below 0°C and that 5 mM imipramine attenuates the temperature effect. These results are explained by supposing that imipramine is able to decrease the transition temperature of the inner mitochondrial membrane lipids as it does for simple lipid systems.     

Differentiation of food vacuolar membranes during endocytosis in Tetrahymena

The origin and differentiation of Tetrahymena pyriformis food vacuolar membranes has been studied by freeze-fracture electron microscopy. By measuring the temperature needed to induce the onset of lipid phase separation (as inferred by the appearance of particle-free regions in replicas) and calculating the changes in average intramembrane particle distribution, a distinct modification of the vacuolar membrane could be observed from the time of its formation from disk-shaped vesicles to its maturation before egestion of its indigestible contents. Whereas the nascent vacuolar membrane first showed signs of phase separation at 9 degrees C, this temperature rose to 14 degrees C in the completed vacuole and then, after lysosomal fusion, eventually declined to 12 degrees C. The average membrane particle density on the PF face increased from 761 +/- 219 to 1,625 +/- 350 per micron 2 during membrane differentiation. Like other membranes of the cell, the vacuolar membrane underwent adaptive changes in its physical properties in cells maintained for several hours at low temperature. This exposure to low temperature caused an equal effect in vacuoles formed before, during, or after the temperature shift-down. Normal changes in the properties of the vacuolar membrane may have some bearing on its programmed sequence of fusion reactions.     

Studies on Acholeplasma laidlawii grown on branched-chain fatty acids

Acholeplasma laidlawii B was grown on the branched-chain fatty acids, 14-methylpentadecanoic acid and 14-methylhexadecanoic acid, and the straight-chain palmitic acid. The incorporation of the branched-chain fatty acids was very effective; more than 90% of the fatty acids of the lipids of this organism consisted of the branched-chain constituents. A somewhat smaller amount (81%) was found in the cells grown with palmitic acid. Differential scanning calorimetry of the isolated membranes showed that distinct lipid phase transitions occurred in between 15 and 31 °C for the 14-methylpentadecanoic acid, 11 and 29 °C for the 14-methylhexadecanoic acid, and 14 and 36 °C for the palmitic acid-enriched membranes. Freeze-fracture electron microscopy showed that the lipid phase transitions were accompanied by particle aggregation only in the case of palmitic acid-enriched membranes. When the branched-chain acid-enriched membranes were quenched from temperatures below the onset of the lipid phase transition, a random distribution of particles on both fracture faces of the membrane was observed. The membranes were incubated with pig pancreatic phospholipase A₂ at various temperatures. Below the onset of the lipid phase transition phosphatidylglycerol was not accessible for this enzyme in palmitate-enriched membranes. However, a fast hydrolysis of 60–75% of the phosphatidylglycerol could be measured in the branched-chain acid-enriched membranes at temperatures below the onset of the lipid phase transition. The residual phosphatidylglycerol could be hydrolyzed at a slower, temperature-dependent rate. The observations show that lipids containing branched-chain acids undergo a cooperative lipid phase transition which does not result in a tight packing of the lipids of the bilayer below the phase transition.     

Modulation of the distribution of plasma membrane intramembranous particles in contact-inhibited and transformed cells.

The intrinsic organization of the plasma membrane differs in normal and transformed cells. With the technique of freeze fracture and electron microscopy contact inhibited 3T3 cells have been shown to contain aggregated plasma membrane intramembranous particles, while transformed cells demonstrate a uniform particle distribution. The distribution of intramembrous particles in transformed cells can be affected by colchicine or vinblastine which induces a dose- and time-dependent particle aggregation. These observations suggest that microtubules and other membrane-associated colchicine-sensitive proteins probably influence the distribution of intrinsic membrane proteins and intramembranous particles in nucleated mammalian cells. An aggregated particle distribution has been observed in 3T3 cells or colchicine-treated transformed cells frozen in media, phosphate-buffered saline or following brief exposure to glycerol, sucrose or dimethyl sulfoxide containing solutions, independent of whether specimens were rapidly frozen from 37 degrees C, room temperature or 4 degrees C incubations. Cells briefly stabilized in 1% formaldehyde yields similar patterns of particle distribution as cells rapidly frozen in media or cryoprotectants. Glutaraldehyde fixation of cells, however, appears to alter the fracturing process in these cells, as visualized by an altered fracture face appearance, decreased numbers of particles, and no particle aggregates. Differences in membrane organization between normal and transformed cells have therefore been demonstrated using a series of preparative methods and colchicine and vinblastine have been shown to modulate intramembranous particle distribution in transformed 3T3 cells.     

Thermotropic lateral translational motion of intramembrane particles in the inner mitochondrial membrane and its inhibition by artificial peripheral proteins

Freeze fracturing and deep etching have been used to study thermotropic lateral translational motion of intramembrane particles and membrane surface anionic groups in the inner mitochondrial membrane. When the inner membrane is equilibrated at low temperature, the fracture faces of both halves of the membrane reveal a lateral separation between intramembrane particles and particle free, large smooth patches. Such separation is completely reversed through free lateral translational diffusion by reversing the temperature. The low temperature induced, particle-free, smooth membrane patches appear to represent regions of protein-excluding, ordered bilayer lipid which form during thermotropic liquid crystalline to gel state phase transitions. When polycationic ferritin is electrostatically bound to anionic groups exposed at the membrane surface at concentrations which inhibit the activities of cytochrome c oxidase and succinate permease, the bound ferritin migrates with intramembrane particles during the thermotropic lateral separation between the membrane particles and smooth patches. When bound polycationic ferritin is cross-bridged with native ferritin, an artificial peripheral protein lattice forms in association with the surface anionic groups and diminishes the thermotropic lateral translational motion of intramembrane particles in the membrane. These results reveal that the anionic groups of metabolically active integral proteins which are known to be exposed at the surface of the inner mitochondrial membrane migrate with intramembrane particles in the plane of the membrane under conditions which induce lipid-protein lateral separations. In addition, cross-bridging of the anionic groups through an artificial peripheral protein lattice appears to diminish such induced lipid protein lateral separations.     

Oxidized phospholipids induce phase separation in lipid vesicles

The thermal behaviour of phospholipid multilamellar vesicles (MLV) made of various molar percentages of DPPC and LPPC, containing also oxidized LPPC (LPPCox), was studied by use of EPR spectroscopy and n-DSPC spin label in order to determine variations in the membrane fluidity brought about by lipid oxidation. Experimental variables were temperature, ranging from 4 to 44 degrees C, and molar percentage composition of DPPC/LPPC/LPPCox ternary mixture. We found that the presence of LPPCox in a percentage higher than both normal phospholipids' heavily hindered membrane formation, while lower percentage of the oxidized lipid with higher DPPC percentages yielded two-components EPR spectra, showing the presence of two different fluidity domains, indicative of membrane phase separation. When LPPC was the dominant lipid in the ternary mixture, simple EPR spectra were observed, indicating homogeneity of MLV membranes. Phase separation observed in the presence of LPPCox was better visible at lower temperature (12 degrees C or less), and almost disappeared with increasing temperature (36 degrees C or more). Furthermore, the correlation time of 16-DSPC in ternary mixture MLVs with higher LPPC percentage (homogeneous membranes) was not affected by the presence of LPPCox, while it normally increased upon DPPC percentage increase, as readily calculated from the EPR spectra featuring simple bands at 24 degrees C. It is concluded that oxidized lipid induces phase separation in more rigid DPPC-rich membranes, while leaving fluidity unaffected in more fluid LPPC-rich membranes, and at higher temperature.     

Use of fluorescence polarization to monitor intracellular membrane changes during temperature acclimation. Correlation with lipid compositional and ultrastructural changes.

Fluorescence polarization of 1,6-diphenylhexatriene (DPH) was used to study the effects of temperature acclimation on Tetrahymena membranes. The physical properties of membrane lipids were found to be highly dependent on cellular growth temperature. DPH polarization in lipids from three different membrane fractions correlated well with earlier freeze-fracture and electron spin resonance observations showing that membrane fluidity progressively decreases in the order microsomes greater than pellicles greater than cilia throughout a wide range of growth temperatures. Changes in membrane lipid fluidity following a shift from high to low growth temperatures proceed rapidly in the microsomes, whereas there is a pronounced lag in the changes of peripheral cell membrane lipids. These data support previous observations that adaptive changes in membrane fluidity proceed via lipid modifications in the endoplasmic reticulum, followed by dissemination of lipid components to other cell membranes. The rapid changes in polarization observed in the microsomal lipids following a temperature shift correspond closely with the time-dependent alterations in both lipid fatty acid composition and freeze-fracture patterns of membrane particle distribution, suggesting that, in the endoplasmic reticulum, lipid phase separation is the primary cause of membrane particle rearrangements.     

Characterization of a low density cytoplasmic membrane subfraction isolated from Escherichia coli

We have used freeze fracture electron microscopy to study the distribution of membrane proteins in the cytoplasmic membrane of Escherichia coli W 3110. While these proteins were distributed randomly at the growth temperature (37 °C), there was extensive protein lipid segregation when the temperature was lowered, resulting in bare patches containing no visible particles (protein), and areas of tightly packed or aggregated particles. To understand the segregation process, we have separated the bare patches from the particle rich membrane areas. Lysis of spheroplasts at 0 °C leads to cytoplasmic membrane fragments with different amounts of membrane particles per unit area; such fragments have been separated on isopycnic sucrose gradients. The bare patches occurred as low density membranes which were completely devoid of particles. They were compared to normal density cytoplasmic membranes with respect to fatty acid composition, protein distribution as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their content of several cytoplasmic membrane marker enzymes.The phospholipid to protein ratio of low density membranes was five times greater than that of normal membranes; unsaturated fatty acids were more abundant in the low density membranes. Most proteins had disappeared from the low density membranes. One protein, which had an apparent molecular weight of 26000 on sodium dodecyl sulfate gels appeared to be concentrated in the low density membranes; it accounted for about 50% of the total protein found in this membrane fraction.Of the cytoplasmic membrane markers tested, NADH oxidase and succinate dehydrogenase were excluded, while d-lactate dehydrogenase remained, and even appeared to be concentrated in the low density membranes.These results indicate that while most membrane proteins are associated with the fluid portion of the bilayer, some proteins evidently associate preferentially with phospholipids in the gel or frozen state.     

Protein-dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing

The hypothesis of a correlation between a 10°–20°C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein-labeled dextran (FD) that is trapped into the membrane. Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12°C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5°C. The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0°C in 5P8 was followed by membrane resealing. This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein-induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine-phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly-L-lysine. Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11°C, which may be due to a gel–liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment. It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin-actin network, may play a major role in membrane resealing.     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes: A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This ‘stiffening’ effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35°C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This corresponds to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10°C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Freeze fracture evidence for lateral phase separations in the plasmalemma of chilling-injured avocado fruit

Summary Unripe avocado fruit (Persea americana Mill. cv Hass) were held at 6 °C either in air or in an atmosphere with 100 PPM ethylene and were assessed for chilling injury after one and two weeks. Injury did not occur in any fruit after one week. After two weeks, the fruit in air were still uninjured, but the fruit subjected to ethylene exhibited chilling injury. When the uninjured fruit (both air-treated for one and two weeks and ethylene-treated for one week) were allowed to warm to room temperature before freezing for freeze fracture electron microscopy, replicas revealed membranes with a randomly dispersed pattern of intramembranous particles (IMPs). However, when these uninjured fruit were frozen for freeze fracture without warming, particle-free domains were visible in the plasmalemma. The membranes of the ethylene-treated, chilling-injured (2 weeks) fruit, on the other hand, contained particle-depleted regions in the plasmalemma of fruit frozen not only from 6 °C but also in those allowed to warm to room temperature before freezing for freeze fracture. These particle depleted microdomains were not seen in fruit kept continuously at room temperature (20 °C), even in the presence of high levels of endogenous ethylene which is produced during normal ripening. We suggest these particle-depleted microdomains formed in the fruit frozen for freeze fracture from low temperatures and in the chilling-injured fruit to be due to lateral phase separations of the membrane components, possibly due to an increase in the viscosity of some membrane lipids, leading to the formation of microdomains of gel phase lipid in the plane of the membrane. These phase separations appear to be initially reversible by raising the temperature, however, this reversibility is apparently lost after injury has occurred. With regard to the cause of chilling injury in avocados, we suggest that some secondary effect is involved due to the long term presence of gel phase lipids in the membrane.     

Low and High Temperature Limits to PSII : A Survey Using trans-Parinaric Acid, Delayed Light Emission, and F(o) Chlorophyll Fluorescence

Many studies have shown that membrane lipids of chilling-sensitive plants begin lateral phase separation (i.e. a minor component begins freezing) at chilling temperatures and that chilling-sensitive plants are often of tropical origin. We tested the hypothesis that membranes of tropical plants begin lateral phase separation at chilling temperatures, and that plants lower the temperature of lateral phase separation as they invade cooler habitats. To do so we studied plant species in one family confined to the tropics (Piperaceae) and in three families with both tropical and temperate representatives (Fabaceae [Leguminosae], Malvaceae, and Solanaceae). We determined lateral phase separation temperatures by measuring the temperature dependence of fluorescence from trans-parinaric acid inserted into liposomes prepared from isolated membrane phospholipids. In all families we detected lateral phase separations at significantly higher temperatures, on average, in species of tropical origin. To test for associated physiological effects we measured the temperature dependence of delayed light emission (DLE) by discs cut from the same leaves used for lipid analysis. We found that the temperature of maximum DLE upon chilling was strongly correlated with lateral phase separation temperatures, but was on average approximately 4°C lower. We also tested the hypothesis that photosystem II (PSII) (the most thermolabile component of photosynthesis) of tropical plants tolerates higher temperatures than PSII of temperate plants, using DLE and F_o chlorophyll fluorescence upon heating to measure the temperature at which PSII thermally denatured. We found little difference between the two groups in PSII denaturation temperature. We also found that the temperature of maximum DLA upon heating was not significantly different from the critical temperature for F_o fluorescence. Our results indicate that plants lowered their membrane freezing temperatures as they radiated from their tropical origins. One interpretation is that the tendency for membranes to begin freezing at chilling temperatures is the primitive condition, which plants corrected as they invaded colder habitats. An alternative is that membranes which freeze at temperatures only slightly lower than the minimum growth temperature confer an advantage.     

Rhombic particle arrays in gill epithelium of a mollusc, Aplysia californica.

Gill epithelia from adult and juvenile Aplysia were examined by conventional thin section and freeze-fracture methods. Freeze-fracture replicas of adult gill epithelium revealed septate and gap junctions, which served as membrane markers for the epithelial cells. In these same cell membranes, non-junctional rhombic arrays of intramembranous particles were observed on prominent ridges on the membrane P fracture face of some epithelial cells. In thin sections of adult epithelium, nerve terminals were observed abutting the lateral plasma membranes near the basal lamina of some epithelial cells. Correlative areas of plasma membrane in freeze-fracture replicas showed a close association between rhombic particle arrays and abutting nerve terminals. In thin sections of juvenile Aplysia, nerve terminals abutting the epithelial cells were not recognizable, and rhombic arrays were not observed in freeze-fracture replicas. This suggested that a developmental association existed between the appearance of rhombic arrays in adult epithelia and their innervation. It is not known with certainty if, in invertebrates, rhombic arrays are an essential structural entity of all innervated cell membranes; however, in the cells thus far studied, there appears to be an associative condition. In the case of the gill epithelium of Aplysia, rhombic arrays are located in the same vicinity as the abutting nerve terminals. Similar arrays of intramembranous particles have been observed in myoneural postjunctional complexes of other invertebrates and have been interpreted to be the morphological expression of neurotransmitter receptors. An analogous explanation is put forth, namely that rhombic arrays may represent the structural correlates of neurotransmitter receptors and/or ionic channels in innervated membranes of invertebrates.     

Effects of lipid-phase separation on the filipin action on membranes of ergosterol-replaced Tetrahymena cells,as studied by freeze-fracture electron microscopy

The effects of lipid-phase separation on the filipin action on pellicle membranes of ergosterol-replaced Tetrahymena pyriformis cells were studied by freeze-fracture electron microscopy. The pellicle membranes with phase separations induced by chilling from 34°C (growth temperature) to lower temperatures (30, 22 and 15°C) were treated with filipin. This produced filipin-induced lesions (“pits”) only in the particulated (liquid) regions along the margin between solid and liquid domains, while they were produced in the particle-free (solid) areas when membranes were chilled to 15°C. The pellicle membranes with lesions induced by filipin at 34°C were chilled to 22°C. This chilling raised larger particle-free areas and more condensed particle-aggregations on the membranes than on the membranes without the filipin treatment. These results suggest that the membrane fluidity affects induction and development of the ergosterol-filipin complex in the membrane.     

The fluidity of chloroplast thylakoid membranes and their constituent lipids: A comparative study by ESR

Comparative measurements were made of the fluidity of chloroplast thylakoids, total membrane lipids and polar lipids utilizing the order parameter and motion of spin labels.No significant differences were found in the fluidity of membranes or total membrane lipids from a wild type and a mutant barley (Hordeum vulgare chlorina f₂ mutant) which lacks chlorophyll $\text{b}$ and a 25 000 dalton thylakoid polypeptide. Redistribution of intrinsic, exoplasmic face (EF) membrane particles by unstacking thylakoid membranes in low salt medium also had no effect on membrane fluidity. However, heating of isolated thylakoids decreased membrane fluidity.The fluidity of vesicles composed of membrane lipids is much greater than that of the corresponding membranes. Fluidity of the membranes, however, increased during greening indicating that the rigidity of the membranes, compared with that of total membrane lipids, is not caused by chlorophyll or its associated peptides. It is concluded that the restriction of motion in the acyl chains in the thylakoids is not caused by chlorophyll or the major intrinsic polypeptide but by some other protein components.     

Lateral phase separations and structural integrity of the inner membrane of rat-liver mitochondria: Effect of compression. Implications in the centrifugation of these organelles

When maintained in the vicinity of the lower transition temperature of their membrane lipids, rat-liver mitochondria undergo lysis as shown by the release of malate dehydrogenase, (an enzyme located within the mitochondrial matrix), in the surrounding medium.Structural changes take place in the membranes of mitochondria subjected to increasing pressure at 0°C, when the pressure reaches 750 kg/cm². Freeze-fracture electron microscopy shows the appearance of smooth areas devoid of particles in fracture faces of mitochondrial membranes, together with zones, where aggregated particles can be seen. Concurrently, a suppression of the malate dehydrogenase structure-linked latency is observed. These structural changes can be prevented by increasing the temperature at which compression is performed. The freeze-etching observations suggest that lateral phase separations occur in mitochondrial membranes subjected to high pressure. This can be explained by supposing that pressure promotes the gel-phase appearance in a lipid system and raises the transition temperature since the transition liquid crystal → gel is accompanied by a decrease in volume. The deterioration of mitochondria subjected to high pressure is interpreted as a result of the lateral phase separation induced by compression in the membranes.These results are discussed with respect to our interpretation of the damaging effects that hydrostatic pressure, generated by centrifugation, exerts on rat-liver mitochondria.     

Thermally induced heterogeneity in microsomal membranes of fatty acid-supplemented Tetrahymena: lipid composition, fluidity and enzyme activity

Thermally induced phase separation was observed to occur in microsomal membranes of the ciliate Tetrahymena pyriformis, using the technique of freeze-fracture electron microscopy. In the present study, we attempted to fractionate the phase-separated membranes which were produced by chilling cells by sucrose density gradient centrifugation. When Tetrahymena was grown in the presence of palmitic acid, cells rapidly incorporated the fatty acid into their phospholipids. The resulting endoplasmic reticulum containing a high level of palmitic acid was more susceptible to thermotropic phase separation. Despite the profound alterations in the fatty acid composition, the cells retained normal growth rate, appearance and cell motility. Smooth microsomes isolated from palmitic acid-supplemented Tetrahymena cells were sonicated and then fractionated into three major subfractions. Fraction-I with lower buoyant density was rich in phospholipids and saturated fatty acids, while Fraction-III with higher density was rather rich in proteins and contained more unsaturated fatty acids in the phospholipids. A significant change was also observed in the polar head composition of phospholipids in these fractions. ESR analysis demonstrated that the extracted lipids from Fraction-III were more fluid than those from Fraction-I. In addition, the motion of the spin probe in the native membranes was more restricted than in extracted lipids. These results indicate that the lipid phase separation causes "squeezing out" of the membrane proteins from the less fluid to the fluid areas. Furthermore, we examined the temperature dependence of the activities of glucose-6-phosphatase and palmitoyl CoA desaturase.     

Studies on Tetrahymena membranes:: Temperature-induced alterations in fatty acid composition of various membrane fractions in Tetrahymena pyriformis and its effect on membrane fluidity as inferred by spin-label study

The fatty acid distribution pattern of lipids extracted from different subcellular components of Tetrahymena pyriformis was found to be significantly different from one type of membrane to another.The growth-temperature shift caused alterations in fatty acid composition. The ratio of palmitoleic to palmitic acid, especially, showed a sharp linear decline with increase of temperature in all of the membrane fractions.The spin labels were rapidly incorporated into Tetrahymena membranes. The order parameter of 5-nitroxide stearate spin label incorporated into various membrane fractions was found to be different for the different membrane fractions, suggesting the following order of the fluidity; microsomes > pellicles > cilia.The fluidity of the surface membranes, cilia and pellicles isolated from Tetrahymena cells grown at 15°C was noticeably higher than that of the membranes from cells grown at 34°C but was not so different with microsomal fractions.The motion of the spin label in the pellicular membrane was more restricted than in its extracted lipids, thus indicating the assumption that in Tetrahymena membranes the proteins influence the fluidity.It was also suggested that a sterol-like triterpenoid compound, tetrahymanol, which is principally localized in the surface membranes, would be involved in the membrane fluidity.     

Spectroscopic and calorimetric studies on trazodone hydrochloride–phosphatidylcholine liposome interactions in the presence and absence of cholesterol

The interaction of antidepressant drug trazodone hydrochloride (TRZ) with dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) in the presence and absence of cholesterol (CHO) was investigated as a function of temperature by using Electron Paramagnetic Resonance (EPR) spin labeling, Fourier Transform Infrared (FTIR) Spectroscopy and Differential Scanning Calorimetry (DSC) techniques. These interactions were also examined for dimyristoyl phosphatidylcholine (DMPC) multilamellar liposomes by using Electron Paramagnetic Resonance (EPR) spin labeling technique. In the EPR spin labeling studies, 5- and 16-doxyl stearic acid (5-DS and 16-DS) spin labels were used to monitor the head group and alkyl chain region of phospholipids respectively. The results indicated that TRZ incorporation causes changes in the physical properties of PC liposomes by decreasing the main phase transition temperature, abolishing the pre-transition, broadening the phase transition profile, and disordering the system around the head group region. The interaction of TRZ with unilamellar (LUV) DPPC liposomes was also examined. The most pronounced effect of TRZ on DPPC LUVs was observed as the further decrease of main phase transition temperature in comparison with DPPC MLVs. The mentioned changes in lipid structure and dynamics caused by TRZ may modulate the biophysical activity of membrane associated receptors and in turn the pharmacological action of TRZ.