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.     

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.     

Membranes of Tetrahymena II. Direct visualization of reversible transitions in biomembrane structure induced by temperature

Rapid temperature changes cause reversible structural transitions in the alveolar membranes of the poikilothermic eukaryote Tetrahymena as revealed by freeze-etch electron microscopy. At an optimal growth temperature of 28°C, 115-Å particles are randomly distributed on the outer faces of the fractured alveolar membranes and apparently corresponding holes are seen on the inner faces. After chilling the cells to 5°C, these particles and holes are largely aggregated. Reheating the cells to 28°C causes a random redistribution of particles and holes. This temperature-induced phenomenon of reversible particle aggregation is discussed with respect to movement of membrane components. We conclude that membrane components move translationally and/or normally to the membrane plane which may be important for transport processes within and across biomembranes.     

Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C₁₈ fatty acids increased in cells grown at lower temperature for all lipid classes, and ω3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the ω3 position of C₁₈ fatty acids increased gradually during a 12-h period after a temperature shift-down to 22°C. However, the mRNA levels of the desA (Δ12 desaturase), desB (ω3 desaturase) and desC (Δ9 desaturase) genes increased within 15 min after a temperature shift-down to 22°C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38°C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22°C and 38°C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.     

The effect of ethanol on the phase behavior of membrane lipids extracted from Clostridium thermocellum strains

The phase behavior of aqueous dispersions of extracted lipids from Clostridium thermocellum wild-type and ethanol-tolerant C919 cells has been examined by DSC. The optimum growth temperature of this anaerobe is 60°C. The wild-type lipids exhibit a broad phase transition centered at 30°C; the C919 mutant lipids show a 10°C lower T_m. The direct addition of growth inhibiting concentrations of ethanol has no significant effect on T_m or headgroup mobility (monitored by ²H-NMR) of either set of lipids. In contrast, wild-type cells adapted to growth in ethanol exhibit a broadened and lower T_m (15–25°C plateau); C919 membrane lipids do not exhibit significantly altered phase behavior when adapted to growth in ethanol. Both wild-type and mutant membranes have fatty acid composition changes upon growth in ethanol, which increases lower-melting components. It is concluded that fatty acid changes which occur upon adaptation of the organism to growth in ethanol are secondary responses and not necessarily direct responses to alter membrane fluidity.     

The relationships between growth temperature,fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37°C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37°C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73–76%), phosphatidylglycerol (9–11%) and cardiolipin (11–13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37°C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at ?13, ?9 and 1°C for membranes from cells grown at 5, 22 and 37°C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5°C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.     

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.     

EFFECTS OF LOWERING TEMPERATURE DURING CULTURE ON THE PRODUCTION OF POLYUNSATURATED FATTY ACIDS IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

The composition of fatty acids and contents of eicosapentaenoic acid (EPA) and polyunsaturated fatty acids (PUFAs) of the economically important marine diatom, Phaeodactylum tricornutum (Bohlin), were investigated to see whether reducing the culture temperature enhances the production of EPA and PUFAs. The contents of EPA and PUFAs of P. tricornutum were found to be higher at lower temperature when cultured at 10, 15, 20, or 25°C. When the cells grown at 25°C were shifted to 20, 15, or 10°C, the contents per dry mass of PUFAs and EPA increased to the maximal values in 48, 24, and 12 h, respectively. The highest yields of PUFAs and EPA per unit dry mass (per unit volume of culture) were 4.9% and 2.6% (12.4 and 6.6 mg·L^?1), respectively, when temperature was shifted from 25 to 10°C for 12 h, both being raised by 120% compared with the control. The representative fatty acids in the total fatty acids, when temperature was lowered from 25 to 10°C, decreased proportionally by about 30% in C_16:0 and 20% in C_16:1(n?7) but increased about 85% in EPA. It was concluded that lowering culture temperature of P. tricornutum could significantly raise the yields of EPA and PUFAs.     

Fatty-Acid Composition of Candida utilis as Affected by Growth Temperature and Dissolved-Oxygen Tension

Analyses were made of the fatty-acid composition of Candida utilis NCYC 321 grown in a chemostat at a dilution rate (equal to growth rate) of 0.1 hr⁻¹ and at temperatures in the range of 30 to 15 C and dissolved oxygen tensions between 75 and <1 mm of Hg. Cells grown under glucose limitation or NH₄⁺ limitation contained mainly C_16:0, C_16:1, C_18:0, C_18:1, C_18:2, and C_18:3 acids as detected by gas-liquid chromatography of methyl esters of the acids from lipids extracted with chloroform-methanol. The relative proportions of these acids varied with the growth temperature and the dissolved-oxygen tension in the culture. A decrease in growth temperature from 30 to 20 C led to an increased synthesis of unsaturated acids in cells grown under either limitation at a fixed-oxygen tension in the range of 75 to 5 mm of Hg. In cultures with a dissolved-oxygen tension of 1 and <1 mm of Hg, a further decrease in temperature to 15 C caused an increased synthesis of unsaturated fatty acids. A decrease in dissolved-oxygen tension led to a diminished synthesis of unsaturated fatty acids in cells grown at a fixed temperature under either limitation. Cells grown at a fixed temperature under glucose limitation synthesized a greater proportion of C₁₆ acids at the expense of C₁₈ acids as the dissolved oxygen tension was decreased from 75 to <1 mm of Hg. A preferential synthesis of C₁₆ acids also occurred as the growth temperature was decreased from 30 to 15 C in cells grown under glucose limitation at a fixed-oxygen tension. The same effect was observed in cells grown under NH₄⁺ limitation when the temperature was lowered from 30 to 20 C; but when the temperature was decreased further to 15 C, the cells synthesized a slightly greater proportion of C₁₈ acids. Synthesis of a large proportion of C₁₆ acids was accompanied by an excretion of pyruvate, and occasionally traces of 2-ketoglutarate, and an increased intracellular accumulation of certain amino acids.     

Fatty acid effects on calcium influx and efflux in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

Low concentrations of fatty acids inhibited initial Ca uptake by sarcoplasmic reticulum vesicles, the extent of inhibition varying with chain length and unsaturation in a series of C₁₄–C₂₀ fatty acids. Oleic acid was a more potent inhibitor of initial Ca uptake than stearic acid at 25°C, whereas at 5°C there was less difference between the inhibitory effects of low concentrations of these fatty acids. When the fatty acids were added later, during the phase of spontaneous Ca release that follows Ca uptake in reactions carried out at 25°C, 1–4 μM oleic and stearic acids caused Ca content to increase. This effect was due to marked inhibition of Ca efflux and slight stimulation of Ca influx. At concentrations of >4 μM, both fatty acids inhibited the Ca influx that occurs during spontaneous Ca release; in the case of oleic acid, this inhibition resembled that of initial Ca uptake at 5°C. The different effects of fatty acids at various times during Ca uptake reactions may be explained in part if alterations in the physical state of the membranes occur during the transition from the phase of initial Ca uptake to that of spontaneous Ca release.     

Homeoviscous theory under pressure. I. The fatty acid composition of Tetrahymena pyriformis NT-1 grown at high pressure

Tetrahymena was grown at up to 260 atm to see if the bilayer-ordering effect of pressure increased the proportion of unsaturated fatty acids in the membrane lipids. Both whole cells and microsomes showed no such change in their fatty acid composition. The most striking effect was seen in the former which showed a pressure-dependent increase in the proportion of C16:0 in relation to C16:Δ9. Homeoviscous adaptation to pressure does not appear to occur in this cell.     

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.     

Transition processes in stratum corneum model lipid membranes with a mixture of free fatty acids

The hydration of model membranes based on ceramide 6 with a mixture of free fatty acids most commonly encountered in the native lipid matrix of stratum corneum, the outermost layer of the mammalian skin, has been studied by neutron diffraction. Membrane hydration with water vapor at a temperature of 25°C is characterized by a small increase in the repeat distance Δd ₀ = 1.0 Å, which is comparable with membrane swelling in the presence of excess water. The kinetics of changes in the repeat distance, connected with an increase of the water layer between bilayers during hydration, and water exchange during the processes of hydration and H-D isotopic substitution, consists of a fast initial and a subsequent slow stage and is well described by exponentials with two characteristic times lying in the range from a few tens of minutes to several hundreds of minutes. During hydration at a temperature of 57°C, the repeat distance increases by Δd ₀ = 1.6 Å, after which the membrane irreversibly separates into two phases. One of the phases is formed mainly by long-chain free fatty acids and is characterized by a large decrease in the repeat distance Δd _ph = 8.3 Å on dehydration. The investigation of the structure of model membranes in the temperature range 20–72°C indicated that the system with 20% (w) of cholesterol in the range of 63–67°C undergoes a structural phase transition caused by the melting of hydrocarbon chains of lipids. In the system with a smaller content of cholesterol, no phase transition was observed up to a temperature of 72°C.     

Induced reversal of order of cell division and DNA replication in Tetrahymena.

The sequence of macronuclear DNA replication (S) and cell division (D) in two normal or synchronized cell cycles in (amicronucleate) Tetrahymena can be symbolized S₁, D₁, S₂, D₂. Using heat shock synchronized cells, the final heat shock (33.8 °C) has been extended from 20 min to 5 h. This leads to an altered sequence: S₁, S₂, D₁, D₂, characterized by reversal of D₁ and S₂. S₂ occurs during the extended shock, D₁ comes in standard time after the shock has been discontinued. Thus prolonged stay at elevated temperature can dissociate two cell cycles into a subcycle of DNA replication followed by a subcycle of cell division. S₁ and S₂ charge the cells with four times the amount of DNA in newly divided cells, and D₁ and D₂ partition this DNA to four cells. Synthesis of DNA between D₁ and D₂ is not required, and it occurs in only few cells.     

Photosynthesis,lipids and proteins in the cyanobacteriumSynechocystis PCC 6803 as affected by temperature

The cyanobacteriumSynechocystis PCC 6803 was grown photoautotrophically in an inorganic medium at constant growth temperatures of 20, 38 (control) or 43°C for 9 h. The up and down-shift of cultivation temperature decreased the growth as measured by culture absorbance and chlorophylla content. However, high temperature slightly increased the oxygen evolution while temperature lower than control inhibited oxygen evolution during the whole incubation period. The protein synthesis studied by¹⁴C-labeled protein declined under low temperature by about 50%. The fatty acid pattern is characterized as lacking in C₂₀/C₂₂ acids but containing large amounts of C₁₆ and C₁₈ polyunsaturated fatty acids, 16:2 and 18:3 in particular. The lower temperature increased the percentage of monounsaturated fatty acids while higher temperature increased the saturated fatty acid content in total lipids and lipid classes studied.     

Studies on Lipolysis of Dairy Lactic Acid Bacteria

Cell-free extracts were prepared by Hughes’ type disintegrator from cells of Lactobacillus casei, L. plantarum, L. helveticus and Streptococcus diacetilactis. Lipolytic activities of these extracts were measured by titration of total acids liberated from tributyrin and butterfal emulsions at given temperature. More acids were liberated from tributyrin than from butterfat, olive oil and composite butter. Lipolytic activities of all extracts for tributyrin were approximately the same at reaction temperature of 30°C, 37°C and 45°C, and were higher at pH 6 to 8. Free fatty acids of C₁₀, C₁₂, C₁₄, C₁₆ and C₁₈ liberated by extracts of L. casei and L. plantarum, were tentatively identified on gas-liquid chromatograms.     

Thermotropic behavior of lipids and the morphology of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> cells with a high content of lysophosphatidylethanolamine

The content of lysophosphatidylethanolamine (LPE) in Y. pseudotuberculosis cells was found to increase during their growth at 8 °C under stationary conditions (without stirring the medium) and at 37°C when the medium contained glucose. The maximum level of LPE (up to 45% of the total phospholipids) was observed in cells grown at 8°C under stationary conditions. Such cells showed decreas growth rate, a reduced yield of biomass, an altered cell morphology, and an increased cell area. The cells contained unsaturated fatty acids, phosphatidylethanolamine (PE), and total phospholipids in small amounts, whereas neutral lipids and diphosphatidylglycerol were abundant. In addition, the cells contained an amount of methylated PE and phospholipids of unknown structure. Irrespective of whether the temperature for growth was low or high, the LPE-rich cells showed a high value (32–36°C) of the maximum temperature of thermal transition of lipids (T _max). This finding is indicative of a densification of the membrane lipid matrix of the LPE-rich cells. The suggestion is made that LPE is accumulated in bacterial cells in response to stress caused by oxygen deficiency and pH decrease in the course of glucose fermentatin. The possible relationship between LPE accumulation and the virulence of Y. pseudotuberculosis cells grown at low temperatures is discussed.     

Membrane phase transitions and the transport of chlortetracycline.

When chlortetracycline is added to a suspension of respiring Staphylococcus aureus cells, the active transport of the antibiotic may be monitored by its fluorescence enhancement as it moves from a polar aqueous environment into the apolar regions of the membrane. The initial rates of transport are temperature dependent with a maximal rate between 35 and 45 °C. Arrhenius plots of the initial rates are biphasic with a transition temperature of 27 °C for control cells. This transition temperature is sensitive to the fatty acid composition of the S. aureus cells. By culturing the cells in the presence of oleic acid or at 10 °C, the S. aureus cells incorporate a larger percentage of unsaturated and branched chain fatty acids into their membranes, resulting in transition temperatures 8–9 °C lower than the control cells. Studies of depolarization of fluorescence also indicate that the mobility of the bound chlortetracycline is temperature-dependent. Temperature transitions occur at the same temperatures as those measured by Arrhenius plots. The transition temperatures indicated by the Arrhenius plots and the polarization studies are believed to reflect order-disorder phase transitions associated with the melting of the phospholipids in the cell envelope.