Correlation between fluidity and fatty acid composition of phospholipid species in Tetrahymena pyriformis during temperature acclimation

The correlation between the fluidity of phospholipids and their fatty acid composition was studied by spin label technique and gas-liquid chromatography for three major phospholipid species in Tetrahymena pyriformis during temperature acclimation. The fluidity of 2-aminoethylphosphonolipid increased within the first 10 h of the cold-acclimation when the content of γ-linolenic acid in 2-aminoethylphosphonolipid was highest, and it then decreased up to 24 h. On the other hand, the fluidities of phosphatidylethanolamine and phosphatidylcholine showed a gradual decrease up to 24 h after the temperature shift, although γ-linolenic acid contents were highest at 10 h after the temperature shift. Thus the fluidity changes of these two phospholipids were interpreted as resulting from the altered content of other fatty acids in addition to γ-linolenic acid, since the γ-linolenic acid content was smaller than that of 2-aminoethylphosphonolipid. The results suggest that the content of γ-linolenic acid in 2-aminoethylphosphonolipid plays a role in regulating the thermal adaptation process.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria Role of cardiolipin in fluidity of mitochondrial membranes

During temperature acclimation of Tetrahymena pyriformis, the changes in fluidity and composition of total lipids from three membrane fractions, mitochondria, pellicles and microsomes were studied by a spin-label technique using a stearate probe and thin-layer and gas-liquid chromatography. The increase of fluidity observed in microsomal and pellicular lipids following the temperature shift from 39 to 15°C corresponds with the increase of the ratio of total unsaturated to saturated fatty acid content. However, despite the increase of this ratio, the fluidity of mitochondrial lipids was found to be constant up to 10 h after the temperature shift. The fluidity of total lipids of mitochondria isolated from Tetrahymena cells grown at 39°C was not changed by removal of cardiolipin, whereas cardiolipin-depleted lipids of mitochondria from 15°C-acclimated cells showed a decrease in fluidity. The re-addition of cardiolipin to the mitochondrial lipids depleted of cardiolipin restored the fluidity to the initial leve, thereby confirming the rigidifying effect of cardiolipin in cold-acclimated cells. These results suggest that cardiolipin may be implicated in maintaining consistent fluidity of mitochondrial membranes against change in thermal environment.     

Modifications of membrane lipid composition following the nutritional shift-up of starved cells a comparison with membrane biogenesis in Tetrahymena

Detailed analyses of lipid composition have been made on various membrane fractions isolated at different intervals after 24 h-starved Tetrahymena cells were refed with nutrient-rich medium. During starvation there was a marked alteration in both phospholipid polar headgroup and acyl chain compositions: an increase in 2-aminoethylphospholipid and γ-linolenic acid (18 : 3) with a concurrent decrease in phosphatidylethanolamine and palmitoleic acid (16 : 1). However, following refeeding, such an altered lipid composition was rather rapidly restored to the initial level of the control cell membranes prior to starvation. This membrane lipid modification was found to occur in good accordance with the recovery of cell size and lipid synthesis. The considerable changes in the principal unsaturated fatty acids, 16 : 1 and 18 : 3, which are formed via the palmitate and stearate desaturation pathways, respectively, were suggested to be accounted for by the levels of desaturases activities. The results of the labeling experiments with radioactive precursors have demonstrated that in the refed cells, there was a more rapid and dynamic transfer or exchange between membranes as compared with that in the exponentially growing control cells. Thus, rapid ameliorative modifications of membrane lipid composition are thought to be required for the urgent growth of membrane systems in the refed cell which should be ready to initiate new division.     

The effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl2 from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which incrased in phosphatidylethanolamine content from 39% (low Na+) to 48% (high Na+) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca2+-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na+-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca2+ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.     

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes

EL4 cells were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membrane phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the $\text{P}$ value of both probes compared to the $\text{P}$ value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, $\text{P}$ values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in $\text{P}$ values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization for monitoring physical properties of membranes (such as ‘fluidity’) is discussed.     

The relatioship between plasma membrane lipid composition and physical-chemical properties II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant difference was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane ‘fluidity’ based on lipid modulation of biological membranes.     

Phospholipid and fatty acid composition of tetrodotoxin receptor-rich membrane fragments from Electrophorus electricus

To study the interaction of voltage-sensitive Na⁺-channels with membrane lipids, the phospholipid and fatty acid composition of highly purified membrane fragments from the remarkably differentiated plasma membrane of Electrophorus electricus has been analyzed. After density gradient fractionation and carrier free electrophoresis, fractions with up to 30 pmol tetrodotoxin binding/mg protein can be obtained, which may correspond to a 50% pure preparation of the extrasynaptic part of the excitable face. Phospholipid classes and cholesterol are separated by one-dimensional thin-layer chromatography in acidic and alkaline solvent systems. The following mean molar contents are found: 40% phosphatidylcholine, 23% phosphatidylserine, 30% phosphatidylethanolamine and 7% sphingomyelin. In a series of 11 animals, significant deviations from these mean values have been observed. The fatty acid composition of the phospholipids has been determined by gas chromatography. Phosphatidylcholine contains more than 50% 16:0, and about 20% unsaturated fatty acids in the C-18 group. Compared to other plasma membrane fractions, this phospholipid is the least differentiated. By contrast, phosphatidylethanolamine and phosphatidylserine show many characteristics in different membrane fractions, especially in their unsaturated components representing more than 50%. 22:6, as the major constituent in these fractions, accounts for a quarter to a third of all fatty acids in these fractions. 18:0 is the main saturated component in these two phospholipids with abundances of typically a quarter or less of all fatty acids. Knowledge of the lipid composition of these excitable membranes may help to conserve binding and structural properties when analyzing lipid-sensitive Na⁺-channels in vitro. It is also useful as a guideline for systematic reconstitution studies.     

Modification of membrane lipids. Phenethyl alcohol-induced alteration of lipid composition in Tetrahymena membranes

Direct measurement of the partition coefficient of $\text{n-}\text{hexane}$ into phosphatidylcholine and phosphatidylcholine-cholesterol bilayers showed that (a) isotropic liquids are not good models for lipid bilayers and (b), Regular Solution Theory cannot, in general, be applied to lipid bilayer membranes at temperatures above their phase transition. Theoretical and experimental evidence is given.     

The polar lipid composition of Mesorhizobium ciceri

The extractable lipid composition of Mesorhizobium ciceri strain HAMBI 1750 grown in a phosphate sufficient medium (79CA) is reported. Cardiolipin (CL—27% of total lipids), phosphatidylglycerol (PG—18%), phosphatidylethanolamine (PE—1%), phosphatidylcholine (PC—30%) and two methylated derivatives of PE, i.e. phosphatidyl-N, N-dimethylethanolamine (DMPE—1%) and phosphatidyl-N-monomethylethanolamine (MMPE—1%), were found to make up the phospholipids of the analysed bacteria. Nonphosphorus, ornithine-containing lipid (OL—10%) was also detected. Polar groups of phospholipids were predominantly acylated with cis-11,12-methyleneoctadecanoyl (lactobacillic) residues, whereas the ornithine lipid contained mainly 3-hexadecanoyloxy-11,12-methyleneoctadecanoic acid bound to the α-amino group.     

The unique lipid composition of gecko (Gekko Gekko) photoreceptor outer segment membranes

This study investigated the lipid and fatty acid composition of gecko photoreceptor outer segment membranes which contain the P521 cone-type pigment. The lipids of gecko photoreceptor outer segment membranes were first extracted and separated by thin layer chromatography (TLC) and then analyzed by gas chromatography (GC). Our results show that gecko photoreceptor outer segment membranes contain less phosphatidylethanolamine (PE) and more phosphatidylcholine (PC) and phosphatidylserine (PS) compared with those of bovine and frog. The content of the polyunsaturated fatty acid, docosahexaenoic acid (DHA), in PC and PS is also the highest yet reported (55 and 63%, respectively). These lipid differences may provide some insight into the specific lipid requirements of cone-type pigments.     

Temperature adaptation of biological membranes. The effects of acclimation temperature on the unsaturation of the main neutral and charged phospholipids in mitochondrial membranes of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the $\text{n-3}\text{and}\text{n-6}$ families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

Control of membrane polar lipid composition in Acholeplasma laidlawii a by the extent of saturated fatty acid synthesis

The low level of endogenous fatty acid synthesis in Acholeplasma laidlawii A strain EF22 was found to be caused by a deficiency of pantetheine in the lipid-depleted growth medium. By supplementing the oleic acid-containing medium with increasing concentrations of pantetheine, saturated fatty acid synthesis was stimulated (having an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 5 μM for pantetheine) and the incorporation of endogenously synthesized fatty acids in membrane lipids increased markedly. Furthermore, carotenoid biosynthesis was stimulated. Exogenous palmitic acid was found to inhibit partially the endogenous fatty acid synthesis. A gradual stimulation of fatty acid synthesis was accompanied by a linear increase in the molar proportion between the two dominating membrane glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol. The total amount of charged membrane lipids decreased upon increasing the degree of fatty acid saturation. These regulations are discussed in terms of membrane stability, and influence of membrane molecular ordering and surface charge density on lipid polar head group synthesis.     

Studies on the size,location and turnover of calcium pools accessible to growing Tetrahymena cells

Tetrahymena pyriformis cells in the logarithmic phase of growth accumulate 2.5–3.75 times as much calcium per unit volume as is present in the growth medium. It appears that most of this calcium is stored in a non-ionic form, with approximately 30% existing in the cilia, near its site of action in effecting ciliary reversal. The exchange of extracellular ⁴⁵Ca²⁺ with the major internal pools is extremely rapid, exhibiting a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of less than 0.5 h. Sites located on the cilia are responsible for 35–50% of Ca²⁺ influx, with the remainder entering through other positions on the cell surface.     

Influence of environmental temperature on mitochondrial membranes

Mitochondrial phospholipids from goldfish lateral line muscle were analysed with respect to polar and apolar groups. Groups of 20 goldfish, acclimated to 5, 20 and 30°C, were used. Temperature-induced shifts of both polar and apolar groups of the mitochondrial phospholipids were observed. The fatty acid composition of mitochondrial phospholipids is characterized by a large amount of polyenoic acids, dominated by docosahexaenoic acid and by octadecadienoic acid. At the higher acclimation temperatures, a significant decrease in docosahexaenoic acid is found. However, the resultant effect of environmental temperature on the degree of unsaturation is small, in contrast to the marked effect on mean chain length. Pronounced changes in the molar ratio of phosphatidylcholine and phosphatidylethanolamine are seen; a decrease in mitochondrial phosphatidylcholine is observed at low acclimation temperature, which is compensated for by a nearly equal increase in phosphatidylethanolamine. The main phospholipids are, apparently, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, comprising 90% of the total pool of 12 species. It is found that the anionic nature of the phospholipids is increased at low acclimation temperatures. We discuss this effect and its probable importance in the stabilization of the surface potential of the mitochondrial membranes.     

The composition and fluidity of normal and leukaemic or lymphomatous lymphocyte plasma membranes in mouse and man

The lymphocyte surface membranes from normal and leukaemic or lymphomatous cells from man and mouse were isolated, characterized, and analyzed both biochemically and by diphenyl hexatriene fluorescence polarization. The cholesterol/phospholipid molar ratio for all the pure lymphocyte plasma membranes was 0.45–0.50, and the fluorescence polarization results showed that values much higher than this were not credible. The lipid composition of all the plasma membranes was remarkably similar, except for the concentration of free fatty acids and glycerides.The latter two were particularly high in the mouse lymphoma membrane and these, rather than a low cholesterol concentration, were responsible for the increased fluidity of the cells.The most prominent protein in most of the plasma membrane preparations was actin. This is found only by some authors, and its presence probably depends on the method of lymphocyte disruption.     

Ethanol-induced changes in the membrane lipid composition of Clostridium thermocellum

When ethanol is added to the growth medium of Clostridium thermocellum ATCC 27405 and C9, a different membrane composition is observed after the period of growth arrest. Changes in fatty acid composition and some unsaturated, branched hydrocarbons have been monitored by GLC-MS. There is a marked increase in normal and anteiso-branched fatty acids at the expense of isobranched fatty acids and an increase in short and unsaturated fatty acids. Thus, an adaptive response to growth in the presence of ethanol induces a membrane containing fatty acids with lower melting points and produces a more ‘fluid’ membrane. The suggestion is made that these membrane changes may be maladaptive to the performance of C. thermocellum.     

Transfer properties of the bovine brain phospholipid transfer protein. Effect of charged phospholipids and of phosphatidylcholine fatty acid composition

The monolayer technique has been used to study the transfer of [¹⁴C]phosphatidylinositol from the monolayer to phosphatidylcholine vesicles. An equivalent transfer rate was found for egg phosphatidylcholine, dioleoylphosphatidylcholine, dielaidoylphosphatidylcholine and dipalmitoylphosphatidylcholine. A reduced transfer rate was found for a shorter-chain derivative, dimyristoylphosphatidylcholine, and for species with two polyunsaturated fatty acid chains such as dilinoleoylphosphatidylcholine, diheptadecadienoylphosphatidylcholine, dilinolenoylphosphatidylcholine and diether and dialkyl derivatives. No activity was found for 1,3-dipalmitoylphosphatidylcholine. The presence of up to 5 mol% phosphatidylinositol in egg phosphatidylcholine vesicles had no effect on the transfer rate. Introduction of more than 5 mol% phosphatidylinositol or phosphatidic acid into the phosphatidylcholine vesicles gradually decreased the rate of phosphatidylinositol transfer from the monolayer. 20 mol% acidic phospholipid was nearly completely inhibitory. Transfer experiments between separate monolayers of phosphatidylcholine and phosphatidylinositol showed that the protein-bound phosphatidylcholine is readily exchanged for phosphatidylinositol, but the protein-bound phosphatidylinositol exchange for phosphatidylcholine occurs at a 20-times lower rate. The release of phosphatidylinositol is dependent on the lipid composition and the concentration of charged lipid in the acceptor membrane, but also on the ratio between donor and acceptor membranes. The main transfer protein from bovine brain which transfer phosphatidylinositol and phosphatidylcholine transfers also phosphatidylglycerol, but not phosphatidylserine or phosphatidic acid. The absence of significant changes in the surface pressure indicate that the phosphatidylinositol and phosphatidylcholine transfer is not accompanied by net mass transfer.     

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 composition and fluidity of adipocyte membranes prepared from young and adult rats

Membranes from adipocytes of adult and young rats have been compared. Phospholipid fatty acids from adult rats were more saturated than those from young rats. This difference was associated with a decreased fluidity in the membranes of the adult rats, which was inferred from measurements of fluorescence polarisation of the fluorescent probe, 1,6-diphenylhexa-1,3,5-triene.     

Osmotic fragility and fluidity of erythrocyte membranes from rats raised on an essential fatty acid deficient diet A spin label study

Erythrocyte membranes from rats raised on a diet with low content of essential fatty acids were studied by osmotic sensitivity tests and spin labeling techniques. This diet induced significant modifications in acylglycerophosphocholine fatty acid composition with regard to 16 : 1, 18 : 1, 18 : 2 (n-6), 20 : 3 (n-9), and 20 : 4 (n-6). No changes in membrane fluidity as monitored by spin label motion were found but the diet caused an increased osmotic sensitivity in essential fatty acid deficient erythrocytes. 50% hemolysis was obtained at a 51.0% dilution of saline with H₂O as compared to a 57.0% dilution for the control material. Membrane fluidity was unaffected by γ-irradiation up to 80 krad.