An X-ray diffraction study on phase transition temperatures of various membranes isolated from Tetrahymena pyriformis cells grown at different temperatures

Mitochondrial, microsomal and pellicular membranes were isolated from Tetrahymena cells grown at 39°C or 15°C, and phospholipids, in turn, were separated from total lipids extracted from these membranes. The effect of growth temperature on their solid-to-fluid phase transition temperature was examined by wide-angle X-ray diffraction. The transition temperatures of phospholipids from mitochondria, microsomes and pellicles were 21, 19 and 26°C for cells grown at 39°C and ?8, ?3 and 6°C for cells grown at 15°C, respectively. All phospholipids were found in a completely fluid state at these growth temperatures. From a comparison between the phospholipids and total lipids from pellicles of cells grown at 39°C, a triterpenoid alcohol, tetrahymanol, caused the transition temperature to increase. The alignment of tetrahymanol in membranes was examined with pellicle's total lipid oriented in a sample holder.     

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.     

Changes in fatty acid distribution and thermotropic properties of phospholipids following phosphatidylcholine depletion in a choline-requiring mutant of Neurospora crassa

Growth of a choline requiring auxotroph of Neurospora crassa on medium lacking exogenous choline produces large changes in the levels of phosphatidylethanolamine and phosphatidylcholine. Whole cell fatty acid distributions were found to vary widely between different phospholipid species of normally growing, choline-supplemented cultures with phosphatidylcholine showing the highest levels of unsaturation and anionic phospholipids and cardiolipin having the lowest. In these lipids, choline deprivation produced little change in fatty acid profiles of phosphatidylethanolamine, whereas changes in fatty acids of phosphatidylcholine and acidic phospholipids resulted in increased levels of unsaturation at both growth temperatures. Microsomal phospholipids also showed fatty acid variability with sharp decreases in phosphatidylcholine unsaturates and increases in acidic phospholipid unsaturated fatty acids at low growth temperatures. Fluorescence polarization of 1,6-diphenylhexatriene in vesicles formed from total cellular and microsomal lipids showed that choline deprivation produces changes in thermotropic properties in the lipids in deprived cultures at either growth temperature. The effective differences in fluorescence polarization between choline-deprived and supplemented cultures grown at a given temperature were found to be comparable to those produced by temperature acclimation in normally growing cultures over a temperature range of 22 K.     

Effects of growth at different temperatures on the physical state of lipids in native microsomal membranes from Tetrahymena

Fluorescence measurements of the probe 1,6-diphenyl-1,3,5-hexatriene in native Tetrahymena pyriformis microsomal membranes revealed characteristic "break points" in curves of polarization vs. temperature. In the 5--35 degree C range, membranes from cells grown at 39 degrees C exhibited two break points, one at 11.6 +/- 0.6 degrees C and another at 23.1 +/- 1.6 degrees C. Membranes from 15 degrees C grown cells also showed two break points, one at 8.0 +/- 1.7 degrees C and another at 17.7 +/- 1.7 degrees C. Complementary measurements of turbidity (absorbance at 360 nm) vs. temperature revealed break points at approximately the same temperatures as observed with the fluorescent probe, thus strengthening the likelihood that the break points signify the onset or termination of lipid phase separations or some other significant structural alteration of lipids. In general, break points measured in the native membrane samples occurred at slightly lower temperatures than did break points in lipids extracted from comparable membranes. This suggests two possible types of protein--lipid interaction. First, there may be a selective withdrawal of relatively highly saturated phospholipid molecular species from the bulk lipid phase and into protein annulus regions. Alternatively, the configuration of the hydrophobic core of certain key membrane proteins may be such that nonspecific interactions with the lipids stabilize the liquid-crystalline phase.     

Factors affecting steroid and prostaglandin secretion by reproductive tissues of cycling and pregnant sows in vitro

Mitochondrial, microsomal and pellicular membranes were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C, and phospholipids, in turn, were separated from total lipids extracted from these membranes. The effect of growth temperature on their solid-to-fluid phase transition temperature was examined by wide-angle X-ray diffraction. The transition temperatures of phospholipids from mitochondria, microsomes and pellicles were 21, 19 and 26 degrees C for cells grown at 39 degrees C and -8, -3 and 6 degrees C for cells grown at 15 degrees C, respectively. All phospholipids were found in a completely fluid state at these growth temperatures. From a comparison between the phospholipids and total lipids from pellicles of cells grown at 39 degrees C, a triterpenoid alcohol, tetrahymanol, caused the transition temperature to increase. The alignment of tetrahymanol in membranes was examined with pellicle'a total lipid oriented in a sample holder.     

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 degrees C after the shift down of growth temperature from 39 degrees 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 degrees C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39 degrees C or 15 degrees C did not change the membrane fluidity (measured at 15 degrees C) of phosphatidylcholine from whole cells grown at 39 degrees C. On the other hand, both cardiolipins of 39 degrees C-grown and 15 degrees C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15 degrees C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15 degrees C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39 degrees C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39 degrees C and 15 degrees C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39 degrees C-grown cells and dipalmitoleoylphosphatidylcholine for 15 degrees 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 fluorescence polarization of 1-acyl-2-cis- or trans-parinaroyl sn-3-glycerophosphorylcholines in model systems and microsomal membranes

Fluorescent lecithin probes containing cis- or trans-parinaric acid (PnA) at the 2-position cis-parinaroylphosphatidylcholine (cis-PnPC) and trans-parinaroyl phosphatidylcholine (trans-PnPC)) showed similar behavior to that of the free cis- or trans-parinaric acids (cis-PnA or trans-PnA) in bilayer vesicles of synthetic saturated lecithins. Transition temperatures detected by cis-PnPc were about 1°C lower than those observed with trans-PnPc. In mixed lecithin vesicles, the trans-PnPc probe monitored a higher temperature melting component than did the cis-probe. Both probes were readily incorporated into microsomal membranes and into sonicated vesicles prepared from the microsomal phospholipids. With either cis- or trans-PnPc no change in polarization ratio was observed for microsomal membranes between 40°C and 0°C but this ratio increased with decreasing temperature between 0°C and ?5°C. However, vesicles of extracted phospholipids showed a continuous increase in polarization ratio with decreasing temperature between 20°C and ?15°C with trans-PnPc and bewteen 5°C and ?15°C with cis-PnPc. These results suggest that the two lecithin probes monitor different environments in the membranes and phospholipid vesicles prepared from them.     

Incorporation of bovine thyroid peroxidase in liposomes

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropiate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (T_c) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37°C, the temperature at which thyroid peroxidase works ‘in vivo’. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.     

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.     

The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa

Experiments were conducted to examine the effects of temperature acclimation on sterol and phospholipid biosynthesis in Neurospora crassa. Cultures grown at high (37 degrees C) and low (15 degrees C) temperatures show significant differences in free and total sterol content, sterol/phospholipid ratios and distribution of major phospholipid species in total lipids and two functionally distinct membrane fractions. The ratio of free sterols to phospholipids in total cellular lipids from 15 degrees C cultures was found to be about one-half that found at 37 degrees C, whereas sterol/phospholipid ratios of mitochondrial and microsomal membranes were found to be higher at the low growth temperature. Total sterol and phospholipid biosynthetic rates showed parallel reductions in cultures acclimating to a shift from 37 to 15 degrees C growth conditions. Distribution of [14C]acetate label into free sterols was significantly lower under these conditions, however; indicating an increase in the conversion rate of sterols to sterol esters at the lower temperature. Mitochondrial and microsomal membrane fractions showed distinct phospholipid distributions which also differed from total lipid distributions at the two growth temperatures. In each case there was a consistent decrease in phosphatidylcholine and a corresponding increase in phosphatidylethanolamine as growth temperatures were lowered.     

A spin-label study of the role of phospholipids in the regulation of membrane-bound microsomal enzymes

The activities of two hepatic microsomal enzymes, glucose 6-phosphatase and UDP-glucuronyltransferase, were determined at assay temperatures in the range between 5 °C and 40 °C. Arrhenius plots of the activities of both enzymes display abrupt changes at about 19 °C. An additional discontinuity at 32 °C is observed in the case of UDP-glucuronyltransferase. Only the latter discontinuity is detected in microsomes subjected to partial treatment with phospholipase A.Lipophilic nitroxide radicals were introduced into samples of the same microsomal preparations and the corresponding electron spin resonance spectra were recorded over the same temperature range. Temperature dependence of an empirical spectral parameter, related to the fluidity of the matrix solubilizing the moleoular probes, reveals apparent breaks at 19 °C and 32 °C in intact microsomes. Only the break at 19 °C was observed in microsomes subjected to sonic disruption. No breaks were detected in plots of data measured in microsomes partially treated with phospholipase A.The correlation between the enzymatic data and the data obtained from lipophilic spin-probes is indicative of the dependence of tightly bound membrane enzymes on the physical state of membrane lipids. The relevance of the data to further studies of the protein-lipid interactions is discussed.     

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.     

The effect of low temperatures on fatty acid composition of crops with different cold resistance

The study was focused on fatty acid (FA) composition of lipids from the seedlings and roots of crops having different cold resistance and grown at 27°C or 4°C. Biosynthesis of FA in the lipids of seedlings and roots of cold-susceptible maize (Zea mays L.) at both growth temperatures was controlled by chloroplast ω6 desaturase and microsomal ω6 desaturase, respectively. The content of linoleic acid was 56.2% and 43.3% in the coleoptiles of maize seedlings grown at 4 and 27°C, respectively, and in the roots it was 52.2% and 38.5%, respectively. The content of α-linolenoic acid in the coleoptiles was 6.7–6.8% at both temperatures, while in the root lipids it was higher at low temperature (3.15% at 27°C vs. 4.7% at 4°C). FA biosynthesis in the seedling coleoptiles of wheat (Triticum aestivum L.) and Siberian wild rye (Elymus sibiricus L.) grown at low temperature was controlled by the chloroplast ω3 desaturase. A minor increase in the content of α-linolenoic acid was observed at low temperature: 29.7% to 30.2% in wheat and 22.8% to 25.8% in wild rye. In the root tissues of these species, the biosynthesis of α-linolenoic acid was controlled by the microsomal ω3 desaturase. The content of α-linolenoic acid was higher at low temperature: in wheat it was 6.1% at 27°C and 17.1%, and 4°C, while in Siberian wild rye, 7.1% and 12.0% at 4 and 27°C, respectively.     

A steady state and differential polarised phase fluorimetric study of the liver microsomal and mitochondrial membranes of thermally acclimated green sunfish (Lepomis cyanellus)

The liver mitochondrial and microsomal membranes of green sunfish and rat were examined by steady state polarisation and differential polarised phase fluorimetry to determine the effects of seasonal adaptation of membrane dynamic structure to temperature. Steady state polarisation studies indicated that the liver mitochondria of green sunfish acclimated to different temperatures showed a greater partial compensation of membrane fluidity for the altered acclimation temperature than did liver microsomal membranes. The fatty acid composition of both membrane preparations generally became more unsaturated at lower acclimation temperatures, though the differences between 5°C and 25°C acclimated fish were more pronounced in the mitochondrial fraction than in the microsomal fraction.Differential polarised phase fluorimetric studies indicated that the rotations of diphenylhexatriene in mitochondrial and microsomal membranes were highly hindered, though the hindrance offered by membranes of 25°C acclimated green sunfish was far greater than that offered by the membranes of 5°C acclimated fish, thus supporting the concept of homeoviscous adaptation. The absolute rotational rate was not consistently affected by acclimation treatment.     

Temperature dependence of the photosynthetic activities in the thylakoid membranes from the blue-green alga Anacystis nidulans

The photosynthetic electron transport and phosphorylation reactions were measured in the room temperature region in the thylakoid membranes prepared from the blue-green alga, Anacystis nidulans. The Arrhenius plot of the Hill reaction with 2,6-dichlorophenolindophenol showed a distinct break of straight lines at 21°C in the membranes from cells grown at 38°C, and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the Hill reaction with ferricyanide showed a break at 13°C in the membranes from cells grown at 38°C, and at 7°C in those from cells grown at 28°C. On the other hand, the Arrhenius plot of the System I reaction with methylviologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system was composed of a straight line in the membranes from cells grown at 28°C as well as at 38°C. The Arrhenius plot of the System II reaction measured by the ferricyanide reduction mediated by silicotungstate in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea also showed a break at 11°C in the membranes from cells grown at 38°C.The Arrhenius plot of the phosphorylation mediated by N-methylphenazonium methylsulfate showed a break at 21°C in the membranes from cells grown at 38°C and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the phosphorylation mediated by the System I reaction showed a break at 24°C in the membranes from cells grown at 38°C.The characteristic features in the Arrhenius plots of the photosynthetic electron transport and phosphorylation reactions are discussed in terms of the transition of physical phase of the thylakoid membrane lipids.     

Changes in thermal phase transition of various membranes during temperature acclimation in Tetrahymena

Changes in the thermal phase transition temperature of membrane lipids were studied by X-ray wide-angle diffraction during adaptation of Tetrahymena pyriformis to a lower growth temperature. After a shift in growth temperature from 39 to 15 degrees C, the phase transition temperature was lowered gradually in microsomal and pellicular phospholipids, whereas that in mitochondrial phospholipids was unchanged for 10 h after the temperature shift. Only a small decrease in the transition temperature of mitochondrial phospholipids was observed, even after 24 h following the shift. Transition temperatures of microsomal, pellicular and mitochondrial phospholipids reached the growth temperature (15 degrees C) about 6, 10 and 24 h after the temperature shift. The temperature dependence of the solid phase in membrane phospholipids was estimated from the 4.2 A peak of the X-ray diffraction pattern. In the case of the phospholipids extracted from cells grown at 39 degrees C, the solid phase was increased upon lowering temperature in a similar manner in all three membrane fractions: mitochondria, pellicles and microsomes. However, in the case of the phospholipids from cells exposed to a lower growth temperature (15 degrees C) for 10 h, the increase in the solid phase was significantly smaller in mitochondrial phospholipids than in two other membrane fractions. The difference in the thermal behaviour of mitochondrial lipid from pellicular and microsomal lipids is discussed in terms of phase transition and phase separation.     

Effects of temperature and growth phase on lipid and biochemical composition of Isochrysis galbana TK1

The lipid and biochemical composition of the haptophyte Isochrysis galbana TK1 was examined. Cultures were grown at 15 °C and 30 °C, and harvested in the exponential and early stationary growth phases. Carbohydrate and protein content varied at the two culture temperatures and growth phases. The highest protein content was found at the exponential growth phase at 15 °C, and the highest carbohydrate content was found at the stationary phase at the same culture temperature. Lipid accumulated in the stationary growth phase and its content was higher at 30 °C than at 15 °C regardless of the growth phase. The neutral lipids were the major class of lipid found in all the cultures. The stationary phase culture had a higher proportion of neutral lipids than the exponential phase culture and the proportion decreased slightly when culture temperature was increased from 15 °C to 30 °C. Phospholipid levels remained constant at the two temperatures, but slightly decreased in the stationary phase. Glycolipids in the exponentially growing cells were higher than those from stationary growth phase and increased with temperature. Polyunsaturated fatty acids (PUFAs) predominated in glycolipids and phospholipids. Cells grown at 15 °C contained higher proportion of 18:3 (n–3) and 22:6 (n–3) with a corresponding decrease in 18:2 (n–6), monounsaturated and saturated fatty acids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rhodnius prolixus LIPOPHORIN: LIPID COMPOSITION AND EFFECT OF HIGH TEMPERATURE ON PHYSIOLOGICAL ROLE

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin‐layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55°C. When it was pretreated at 75°C, maximal inhibition of phospholipid transfer was observed after 3‐min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75°C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55°C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75°C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65°C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85°C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.     

Temperature-induced modifications of glycosphingolipids in plasma membranes of Neurospora crassa

Plasma membranes isolated from a cell-wall-less mutant of Neurospora crassa grown at 37 and 15°C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37°C membranes, while 15°C plasma membranes exhibited a ratio of nearly 2.0. Membranes formed under both growth conditions were found to contain glycosphingolipids. Cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolipids and a corresponding 2-fold reduction of phospholipid levels. The high glycosphingolipid content at 15°C compensates for the reduced levels of phospholipids in such a way that sterol/polar lipid ratios are almost the same in plasma membranes under the two growth conditions. Temperature-dependent changes in plasma-membrane phospholipid and glycosphingolipid species were also observed. Phosphatidylethanolamine levels were sharply reduced at 15°C, in addition to a moderate increase in levels of unsaturated phospholipid fatty acids. Glycosphingolipids contained high levels of long-chain hydroxy fatty acids, which constituted 75% of the total fraction at 37°C, but only 50% at 15°C. Compositional changes were also observed in the long-chain base component of glycosphingolipids with respect to growth temperature. Fluorescence polarization studies indicate that the observed lipid modifications in 15°C plasma membranes act to modulate bulk fluidity of the plasma-membrane lipids with respect to growth temperature. These studies suggest that coordinate modulation of glycosphingolipid, phospholipid and sterol content may be involved in regulation of plasma-membrane fluid properties during temperature acclimation.     

Possible involvement of acetyl coenzyme A carboxylase as well as fatty acid synthetase in the temperature-controlled synthesis of fatty acids in Saccharomyces cerevisiae

Fatty acid synthetase (FAS) preparations from Saccharomyces cerevisiae cells grown at either 35 or 10 degrees C produced the same products at different temperatures and showed quite similar temperature-dependencies in Arrhenius plots, with break points at 25 degrees C. This break point does not appear to reflect a phase transition of phospholipids present in the purified FAS preparations but rather is associated with protein conformational changes. S. cerevisiae cells grown at 35 degrees C and then shifted to 10 degrees C produced fatty acids with a shorter average chain length than those fatty acids synthesized at 10 degrees C by cells already adapted to 10 degrees C (hyper response). Acetyl-CoA carboxylase activity was relatively higher in the cells grown at 35 degrees C than in the cells grown at 10 degrees C; moreover, fatty acids with longer average chain lengths were synthesized in vitro at higher malonyl-CoA concentrations, which was consistent with the difference in the average chain lengths of newly synthesized fatty acids in cells grown at 35 and 10 degrees C. However, the activity levels of acetyl-CoA carboxylase and fatty acid synthetase alone did not account for the hyper response phenomena.