A 2H-NMR study of Acholeplasma laidlawii membranes highly enriched in myristic acid

Myristic acid specifically deuterated at several positions along the acyl chain was biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B to the level of ?90%. ²H-NMR was used to study the molecular order and lipid phase composition of the membranes as a function of temperature. Isolated membranes and intact cells give rise to similar ²H spectra. Below 25°C the spectra exhibit a broad gel phase component which at 0°C reaches the rigid limit value expected for an immobilized methylene group. Spectral moments were used to determine the relative amounts of gel and liquid crystalline phase lipids throughout the gel-liquid crystal phase transition. The results indicate that at the growth temperature (37 or 30°C) the A. laidlawii B membrane lipids are ～85–90% in the gel state, and that protein has little effect on lipid order of the liquid crystalline lipid, but leads to an increase in the linewidth by approx. 20%.     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43°C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20°C, but in a liquid crystalline state when cells were grown at 37 and 43°C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

A 31P- and 2H-NMR study on lecithins in liquid crystalline polyoxyethylene detergents

Phosphatidylcholines were incorporated into hexagonal liquid cyrstalline mixtures of the non-ionic detergents Triton X-100 and octaethyleneglycoldodecylether with D₂O. It is shown by nuclear magnetic resonance (NMR) that the phospholipids adopt the hexagonal liquid crystalline structure of the detergent host lattice. The anisotropic motion of the phospholipid headgroups seems to be unaffected, whereas the acyl chains are disordered. Increasing phospholipid concentration leads to separation of a lamellar phase. The lamellar structure is also preferred at elevated temperatures. Phosphatidylcholines with saturated acyl chains undergo a transition from the hexagonal liquid crystalline to an ordered lamellar state. The shape of the ³¹P-NMR signals suggests that pure gel phase phospholipid separates out. The headgroup region of this gel phase phospholipid becomes immobilized after a few weeks of storage below the transition temperature as judged from ³¹P-NMR. At the same time ²H-NMR exhibits a new signal from D₆₂O undergoing slow isotropic motion. This behavior bears resemblance to the formation of a coagel in fatty acid-water systems.     

Effects of lipid composition on the membrane activity and lipid phase behaviour of Vibrio sp. DSM14379 cells grown at various NaCl concentrations

The membrane lipid composition of living cells generally adjusts to the prevailing environmental and physiological conditions. In this study, membrane activity and lipid composition of the Gram-negative bacterium Vibrio sp. DSM14379, grown aerobically in a peptone-yeast extract medium supplemented with 0.5, 1.76, 3, 5 or 10% (w/v) NaCl, was determined. The ability of the membrane to reduce a spin label was studied by EPR spectroscopy under different salt concentrations in cell suspensions labeled with TEMPON. For lipid composition studies, cells were harvested in a late exponential phase and lipids were extracted with chloroform-methanol-water, 1:2:0.8 (v/v). The lipid polar head group and acyl chain compositions were determined by thin-layer and gas-liquid chromatographies. ³¹P-NMR spectroscopy was used to study the phase behaviour of the cell lipid extracts with 20 wt.% water contents in a temperature range from −10 to 50 °C. The results indicate that the ability of the membrane to reduce the spin label was highest at optimal salt concentrations. The composition of both polar head groups and acyl chains changed markedly with increasing salinity. The fractions of 16:0, 16:1 and 18:0 acyl chains increased while the fraction of 18:1 acyl chains decreased with increasing salinity. The phosphatidylethanolamine fraction correlated inversely with the lysophosphatidylethanolamine fraction, with phosphatidylethanolamine exhibiting a minimum, and lysophosphatidylethanolamine a maximum, at the optimum growth rate. The fraction of lysophosphatidylethanolamine was surprisingly high in the lipid extracts. This lipid can form normal micellar and hexagonal phases and it was found that all lipid extracts form a mixture of lamellar and normal isotropic liquid crystalline phases. This is an anomalous behaviour since the nonlamellar phases formed by total lipid extracts are generally of the reversed type.     

Effect of temperature on the NMR spectra of sonicated dispersions of isomers of dipalmitoylphosphatidylcholine

Sonicated dispersions of 1,2-dipalmitoylsn-glycero-3-phosphorylcholine and of 1,3-dipalmitoylglycero-2-phosphorylcholine were examined by proton nuclear magnetic resonance (NMR) as a function of temperature. The —(CH₂)_n)— peak in the spectrum of the sn-3-isomer of dipalmitoylphosphatidylcholine showed the characteristic dramatic changes in the peak intensity and width associated with the phase transition between the liquid crystalline and gel states of the phospholipid. This occurred over a 2–3°C temperature range with the midpoint of the transition at 38.5°C. With the 2-isomer the change in phase took place over a similar temperature range but the midpoint was at 33.8°C. This lower phase transition temperature is presumably the result of increased acyl chain mobility caused by the increased separation of the two acyl chains by the centre carbon of the glycerol backbone. The effect of sonication of the broadening of the range and lowering of the midpoint temperature of the phase transition from that of the corresponding unsonicated dispersions was similar with each isomer. This suggests that the overall geometry of the sonicated vesicles of the isomers is similar.     

Phase behavior of the major lipids of Tetrahymena ciliary membranes

The major lipids of Tetrahymena membranes have been purified by thin-layer and high pressure liquid chromatography and the phosphatidylethanolamine and aminoethylphosphonate lipids were examined in detail. ³¹P-NMR, X-ray diffraction and freeze-fracture electron microscopy were employed to describe the phase behavior of these lipids. The phosphatidylethanolamine was found to form a hexagonal phase above 10°C. The aminoethylphosphonate formed a lamellar phase up to 20°C, but converted to a hexagonal phase structure at 40°C. Small amounts of phosphatidylcholine stabilized the lamellar phase for the aminoethylphosphonate. ³¹P-NMR spectra of the intact ciliary membranes were consistent with a phospholipid bilayer at 30°C, suggesting that phosphatidylcholine in the membrane stabilized the lamellar form, even though most of the lipid of that membrane prefers a hexagonal phase in pure form at 30°C. ³¹P-NMR spectra also showed a distinctive difference in the chemical shift tensor of the aminoethylphosphonolipid, when compared to that of phosphatidylethanolamine, due to the difference in chemical structure of the polar headgroups of the two lipids.     

Synthesis and physical properties of phosphatidylcholines containing ω-cyclohexyl fatty acyl chains

Phosphatidylcholines (PCs) with cyclohexyl fatty acids as acyl chains were synthesized from 11-cyclohexyl and 13-cyclohexyl fatty acids and their physical properties were examined. The thermotropic behavior and barrier function of liposomal membranes formed from these PCs were studied. These PCs showed about 10°C lower gel-to-liquid crystalline phase transition temperatures (T_c) than the corresponding straight-chain PCs. The properties of mixtures of these cyclohexyl acyl PCs with straight-chain PCs were rather different from those observed with mixtures of straight-chain PCs. Cyclohexyl fatty acyl PCs showed barrier functions even above the T_c unlike the corresponding straight-chain PCs. These results indicate significant differences between the overall packing of cyclohexyl fatty acyl PCs and of the corresponding straight-chain PCs both in the gel state and in the liquid crystalline state. The significance of these cyclohexyl acyl chains in polar lipids, which are abundant in the thermophilic acidophilic bacterium, Bacillus acidocaldarius, is discussed.     

The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.     

Phase behavior of ethanolamine plasmalogen

In the present study the phase behavior of multilamellar dispersions of 1-O-(1′-alkenyl)-2-oleoyl-glycerophosphoethanolamine (ethanolamine plasmalogen), 1-O-alkyl-2-oleoyl-glycerophosphoethanolamine and 1-acyl-2-oleoyl-glycerophosphoethanolamine was compared using differential scanning calorimetry (DSC) and ³¹P-NMR. The three compounds differed only in the type of bonding (vinyl ether, alkyl ether or acyl ester) linking the aliphatic moiety to position 1 of sn-glycerol.The gel to liquid-crystalline phase transition temperature as determined by DSC was lowest for ethanolamine plasmalogen (26°C) and was similar for the alkylacyl and diacyl analogs (29.5° and 30°C, respectively). Enthalpies of the G → L phase transition were not significantly different for the three phospholipids tested.Ethanolamine plasmalogen undergoes the lamellar to hexagonal phase transition at 30°C, the analogous alkylacyl-glycerophosphoethanolamine(alkylacyl-GPE) and diacyl-GPE at 53°C and 69°C, respectively. Thus, an alkenyl ether bond in position 1 of sn-glycerol, the structural characteristic of plasmalogens, effectively stabilizes the hexagonal H_II arrangement of ethanolamine glycerophospholipids, while it has relatively little effect on destabilization of the lamellar gel state.     

A variety of glycolipids in green photosynthetic bacteria

The compositions of glycolipids in the following seven strains of green photosynthetic bacteria were investigated at the molecular level using LC–MS coupled with an evaporative light scattering detector: Chlorobium (Chl.) limicola strains Larsen (30 °C as the optimal cultivation temperature) and DSM245 (30 °C), Chlorobaculum (Cba.) tepidum strain ATCC49652 (45 °C), Cba. parvum strain NCIB8327 (30 °C), Cba. limnaeum strain 1549 (30 °C), Chl. phaeovibrioides DSM269 (30 °C), and Chloroflexus (Cfl.) aurantiacus strain J-10-fl (55 °C). Dependence of the molecular structures of glycolipids including the chain-length of their acyl groups upon bacterial cultivation temperatures was clearly observed. The organisms with their optimal temperatures of 30, 45, and 55 °C dominantly accumulated glycolipids possessing the acyl chains in the range of C₁₅–C₁₆, C₁₆–C₁₇, and C₁₈–C₂₀, respectively. Cba. tepidum with an optimal temperature of 45 °C preferred the insertion of a methylene group to produce finally a C₁₇-cyclopropane chain. Cfl. aurantiacus cultured optimally at 55 °C caused a drastic increase in the chain-length. Notably, the length of such acyl groups corresponded to that of the esterifying chain in the 17-propionate residues of self-aggregative bacteriochlorophylls-c/d/e, indicating stabilization of their supramolecular structures through hydrophobic interactions among those hydrocarbon chains. Based on the detailed compositions of glycolipids, a survival strategy of green photosynthetic bacteria grown in the wide range of temperatures is discussed.     

The effects of hydration and divalent cations on lamellar-nonlamellar phase transitions in membranes and total lipid extracts from Acholeplasma laidlawii A-EF22 – a 2H NMR study

Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with 75 μm α-deuterated palmitic acid (16:0-d ₂) and 75 μm α-deuterated oleic acid (18:1c-d ₂), or with 150 μm 18:1c-d ₂. The fatty acids were incorporated into the membrane lipids and ²H NMR spectra were recorded from intact membranes, total lipid extracts, and the combined glucolipid and neutral lipid fractions of a total lipid extract. The lipids in intact membranes form a bilayer structure up to at least 70 °C. The same result was obtained with membranes digested with pronase, which removes a large fraction of the membrane proteins. A reversed hexagonal liquid crystalline (H_II) phase was formed below 70 °C by the total lipid extracts hydrated with 20 and 30% (w/w) water; in the presence of 40% (w/w) water only one of the extracts formed an H_II phase below 70 °C. The H_II phase was formed at higher temperatures with an increasing water content. However, only a lamellar liquid crystalline (L _α ) phase was formed up to 70 °C by the total lipid extracts when the water concentrations were 50% (w/w) or higher. The temperature (T _LH) for the L _α to H_II phase transition in the combined glucolipid and neutral lipid fractions was only 2–3 °C lower than for the total lipids, and the phospholipids thus have a very modest influence on the T _LH value. Physiologically relevant concentrations of Ca²⁺ and Mg²⁺ ions did not affect the phase equilibria of total lipid extracts significantly. It is concluded from comparison with published data that the membrane lipids of the cell wall-less bacterium A. laidlawii have a smaller tendency to form reversed nonlamellar phases than the membrane lipids of three bacterial species surrounded by a cell wall. Received: 10 March 1997 / Accepted: 4 July 1997     

Interaction of melittin with dimyristoyl phosphatidylcholine liposomes. Evidence for boundary lipid by raman spectroscopy

The interaction of melittin, a polypeptide consisting of 26 amino acid residues, with dimyristoyl phosphatidylcholine bilayers was investigated by vibrational Raman spectroscopy. Spectral peak height intensity ratios, involving vibrational transitions in both the 3000 cm^?1 acyl chain methylene carbon-hydrogen stretching mode region and the 1100 cm^?1 acyl chain carbon-carbon skeletal stretching mode interval, served as temperature profile indices for monitoring the bilayer order-disorder processes. For a lipid : melittin molar ratio of 14 : 1 two order-disorder transitions were observed. In comparison to a gel to liquid crystalline phase transition of 22.5°C for the pure lipid, the lower transition, exhibiting a 2°C width, is centered at 17°C and is associated with a depression of the main lipid phase transition of dimyristoyl phosphatidylcholine. The second thermal transition, displaying a 7°C interval, occurs at approx. 29°C and is associated with the melting behavior of approximately seven immobilized boundary lipids which surround the inserted hydrophobic segment of the polypeptide. For a lipid : melittin molar ratio of 10 : 1 two thermal transitions are also observed at 11 and 30°C. As before, they represent, respectively, the main gel to liquid crystalline phase transition and the melting behavior of approximately four boundary lipids attached to melittin. From these data alternative schemes are suggested for disposing the immobilized lipids around the hydrophobic portion of the polypeptide within the bilayer.     

Sterol-phospholipid interactions in model membranes Effect of polar group substitutions in the cholesterol side-chain at C20 and C22

The interactions of phospholipids with four different cholesterol derivatives substituted with one OH or one keto group at position C₂₀ or C₂₂ of the side-chain were studied. The derivatives were the 22,R-hydroxy; 22,S-hydroxy; 22-keto- and 20,S-hydroxycholesterol. Two aspects of the interactions were investigated: (1) the effect of the cholesterol derivatives on the gel → liquid crystalline phase transition of dipalmitoylphosphatidylcholine (DPPC) and of dielaidoylphosphatidylethanolamine (DEPE) monitored by differential scanning calorimetry and (2) The effect on the lamellar → hexagonal H_II phase transition of DEPE monitored by DSC and by ³¹P-NMR to determine structural changes. The gel → liquid crystalline phase transition was affected by the cholesterol derivatives to a much larger extent in the case of DPPC than of DEPE. In both cases, there was a differential effect of the four derivatives, the 22,R-hydroxycholesterol being the less effective. In DPPC-sterol 1:1 systems, 22,R-hydroxycholesterol does not suppress the melting transition, the ΔH values becomes 7.1 kcal · mol^?1 as compared to 8.2 kcal · mol^?1 for the pure lipid. 22,S-OH cholesterol has a much stronger effect (ΔH = 3.1 kcal · mol^?1) and 22-ketocholesterol suppresses the transition completely. In DEPE mixtures of all these compounds, the melting transition of the phospholipid is still observable. The transition temperature was shifted to lower values (?13.5°C in the presence of 20,S-OH cholesterol). The ΔH of the transition was lowered by these compounds except in DEPE-22,R-OH cholesterol mixtures and the cooperativity of the transition (reflected by the width at half peak height) was reduced. The lamellar → hexagonal H_II phase transition was also affected by the presence of these cholesterol derivatives. The transition temperature value was depressed with all these compounds. 20,S-OH cholesterol was the most effective followed by 22,R-OH cholesterol. The ΔH of the transition was not strongly affected. The molecular interfacial properties of these derivatives were studied by the monomolecular film technique. It is most likely that 22,R-OH cholesterol due to the hydroxyl groups at the 3β- and 22,R-positions orients with the sterol nucleus lying flat at the air/water interface, since the compression isotherm of either the pure sterol or the DOPC-sterol mixture (molar ration, 1:1) monomolecular film exhibits a transition at approx. 103 Ų, corresponding to the area of revolution of the sterol nucleus. This remarkable property, due probably to the existence of a kink between the side-chain and the long axis of the steroid nucleus, might explain the smaller effect of this sterol on the melting transition of either PC or PE systems.     

Thermodesulfobacterium geofontis sp. nov., a hyperthermophilic, sulfate-reducing bacterium isolated from Obsidian Pool, Yellowstone National Park

A novel sulfate-reducing bacterium designated OPF15^T was isolated from Obsidian Pool, Yellowstone National Park, Wyoming. The phylogeny of 16S rRNA and functional genes (dsrAB) placed the organism within the family Thermodesulfobacteriaceae. The organism displayed hyperthermophilic temperature requirements for growth with a range of 70–90 °C and an optimum of 83 °C. Optimal pH was around 6.5–7.0 and the organism required the presence of H₂ or formate as an electron donor and CO₂ as a carbon source. Electron acceptors supporting growth included sulfate, thiosulfate, and elemental sulfur. Lactate, acetate, pyruvate, benzoate, oleic acid, and ethanol did not serve as electron donors. Membrane lipid analysis revealed diacyl glycerols and acyl/ether glycerols which ranged from C_14:0 to C_20:0. Alkyl chains present in acyl/ether and diether glycerol lipids ranged from C_16:0 to C_18:0. Straight, iso- and anteiso-configurations were found for all lipid types. The presence of OPF15^T was also shown to increase cellulose consumption during co-cultivation with Caldicellulosiruptor obsidiansis, a fermentative, cellulolytic extreme thermophile isolated from the same environment. On the basis of phylogenetic, phenotypic, and structural analyses, Thermodesulfobacterium geofontis sp. nov. is proposed as a new species with OPF15^T representing the type strain.     

Vibrational raman spectra of lipid systems containing amphotericin B

Resonance-enhanced and normal vibrational Raman spectra were observed for both multilamellar and single-wall vesicle assemblies of dimyristoyl phosphatidylcholine containing amphotericin B, a channel-forming polyene antibiotic, and cholesterol. The decrease in the frequency of the polyene antibiotic C  C stretching mode at 1556 cm^?1 and the increase in intensity of the CCH in-plane deformation mode at 1002 cm^?1 indicate that amphotericin B is ordered in a lipid-cholesterol medium similarly to the solid, but is surrounded by a slightly more polar environment. The intensity of the C  C stretching mode I₁₅₅₆ decreases 4-fold during the broadened gel to liquid crystalline phase transition (16–32°C) of dimyristoyl lecithin-cholesterol (4 : 1) multilayers. Other resonance-enhanced vibrations of amphotericin B exhibit similar behavior. For amphotericin B in pure dimyristoyl lecithin multilayer or vesicle systems, however, the vibrational intensity associated with the C  C stretching mode remains constant during the melting of lipid hydrocarbon chains. In addition, a third effect occurs in liquid crystalline egg lecithin-cholesterol (4 : 1, mol ratio) multilayers in which I₁₅₅₆ first increases by 25% between 3 and 25°C, in parallel with the loss of active channels, and then remains constant as the temperature increases from 25 to 42°C. This latter intensity pattern is masked in the dimyristoyl lecithin-cholesterol system by the overwhelming effect upon the C  C mode from changes in the lipid chain packing characteristics which occur during the phase transition.The broadened phase transition in 4 : 1 dimyristoyl lecithin-cholesterol multilayers (16–32°C), as followed by the ratio of intensities at 2880 and 2850 cm^?1 (asymmetric and symmetric methylene C-H stretching modes, respectively) is slightly narrowed by the addition of amphotericin B, and effect from which a binding stoichiometry at 24° of 1 : 1 amphotericin B : cholesterol is estimated. This stoichiometry was confirmed by differential calorimetric scans, which also show the presence of a peak proportional to cholesterol content.Raman I_2880/2850 peak height ratios in pure dimyristoyl lecithin bilayers were increased over the 14–38°C range by amphotericin B, a spectral effect which suggests an ordering of the lipid matrix perhaps as a consequence of the polyene binding to the bilayer surface. For bilayers containing cholesterol, the ratios of intensities of the 2935 cm^?1 feature, composed mainly of acyl chain terminal methyl and underlying methylene C-H stretching modes, to the 2850 cm^?1 feature are significantly increased by amphotericin B. This effect indicates that the antibiotic penetrates the bilayer in the lipid-sterol system.     

The temperature-mediated metabolism of 1-acyl-lysophosphatidyl glycerol in cerulenin-treated cultures of Bacillus megaterium.

When [U-¹⁴C]palmitate was added to a culture of $\text{B. megaterium}$ that had been grown at 35°, transferred to 20° and treated with cerulenin, label was initially incorporated into lysophosphatidyl glycerol. The labeled lyso derivative, in turn, was converted to phosphatidyl glycerol, apparently by esterification of the 2-position with endogenous acyl groups. Labeled lysophosphatidyl glycerol synthesis at 20° was observed only when a culture was treated with cerulenin prior to the addition of [U-¹⁴C]palmitate. When [U-¹⁴C]palmitate was added before cerulenin, labeled lysophosphatidyl glycerol formation was not detected. When chloramphenicol was added with cerulenin at the time of culture transfer from 35° to 20°, the synthesis of lysophosphatidyl glycerol was unaffected but the rate of its esterification to phosphatidyl glycerol was significantly retarded. Transfer of such a culture back to 35° resulted in a marked acceleration in the rate of conversion of lysophosphatidyl glycerol to phosphatidyl glycerol.     

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

Lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii B

The thermotropic behaviour of fatty acid-homogeneous membranes of Acholeplasma laidlawii B was investigated by Fourier transform infrared spectroscopy. The organism was grown at 37°C in the presence of avidin, an inhibitor of fatty acid synthesis, in a medium supplemented with pentadecanoic acid-d₂₉; the enrichment of the membranes with this fatty acid was 95%. The temperature-dependent phase behaviour of the membranes was studied via the C–D stretching vibrational modes of the membrane lipids and was compared with that of the lipid extract. The high level of fatty acid homogeneity results in a sharp (for natural membranes) gel to liquid crystalline phase transition. The transition, in both the membranes and extracted lipids, is centered at about 6°C above the growth temperature. During the transition two principal liquid states are evident, one being more conformationally ordered than the other. The effect of proteins on the principal lipid phase transition is minimal. However, in the intact membranes there is evident a weaker, lower temperature transition, which is not evident in the extracted lipids.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385–392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15°C after the shift down of growth temperature from 39°C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15°C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39°C or 15°C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39°C or 15°C did not change the membrane fluidity (measured at 15°C) of phosphatidylcholine from whole cells grown at 39°C. On the other hand, both cardiolipins of 39°C-grown and 15°C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15°C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15°C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39°C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39°C and 15°C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39°C-grown cells and dipalmitoleoylphosphatidylcholine for 15°C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.