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 gamma-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 gamma-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 gamma-linolenic acid, since the gamma-linolenic acid content was smaller than that of 2-aminoethylphosphonolipid. The results suggest that the content of gamma-linolenic acid in 2-aminoethylphosphonolipid plays a role in regulating the thermal adaptation process.     

Studies in Tetrahymena membranes substrates for desaturation of fatty acyl chains in Tetrahymena pyriformis microsomes

[1-14-C]Palmitoyl-Co A was incubated with Tetrahymena microsomes containing the complete enzyme system for desaturation during various time periods. The level of [1-14C]palmitoleoyl-CoA increased to a maximum during the 1--3 min incubation time, while [1-14C]palmitoleic acid in the phospholipid reached a maximum level during 6--7 min incubation time. The radioactivity of [1-14C]palmitoleic acid in free fatty acid and the triglyceride fraction was not significantly observed upon 3 min incubation. Incubation of [1-14C]palmitoyl-CoA with microsomes in the absence of NADH produced [1-14C]palmitoyl lipid without desaturation. Radioactive palmitic acids in the microsomal lipids were not converted to palmitoleic acids after addition of NADH by the complete enzyme system. When microsomes prepared from cells labeled with [1-14C]palmitic acid or [1-14C]stearic acid were incubated alone in the presence of O2 and NADH, no significant increase in [1-14C]palmitoleic acid in the phospholipid was observed, wherease an increase in [1-14C]linoleic acid and gamma-[1-14C]linolenic acid did occur at the expense of [1-14C]oleic acid in the phospholipid. From these results it can be concluded that the enzyme involving desaturation of palmitic acid to palmitoleic acid requires palmitoyl-CoA as the substrate. However, the possibility of oleoyl and linoleoyl phospholipids being substrates in the desaturation of Tetrahymena microsomes was suggested.     

Self-regulation of membrane fluidity. The effect of saturated normal and methoxy fatty acid supplementation on Tetrahymena membrane physical properties and lipid composition.

Tetrahymena cells elongated and desaturated massive supplements of palmitic or lauric acid at nearly twice the rates employed by unfed cells, thereby maintaining constant the physical properties of their membrane lipids. However, when a mixture of the 9- and 10-monomethoxy derivatives of stearic acid was administered, these compounds were incorporated without further metabolism. The marked fluidizing effect of the phospholipid-bound methoxy-fatty acids elicited an immediate reduction in fatty acid desaturase activity, the pattern of change being very similar to that induced by supplements of polyunsaturated fatty acids. The modulation of fatty acid desaturase activity by methoxy-acids clearly seems to be governed by membrane fluidity rather than by some form of end product inhibition of the type which might have been postulated to explain the similar effect caused by polyunsaturated fatty acids.     

Temperature-induced changes in fatty acid unsaturation of Tetrahymena membranes do not require induced fatty acid desaturase synthesis.

The usual rise in phospholipid-bound palmitoleic acid of Tetrahymena pyriformis cells slowly acclimating to low temperature exposure can be prevented by cycloheximide. This reduction in fatty acid desaturation is not caused by specific inhibition of a temperature-induced synthesis of a fatty acid desaturase but rather by a general effect equally conspicuous in isothermal cells. Cycloheximide-inhibited cells chilled and analyzed quickly, before long term ill effects of the drug are expressed, exhibit the rise in unsaturated fatty acids typical of temperature-acclimating cells.     

The effect of temperature on unsaturated fatty acid loss in Tetrahymena pyriformis.

Cultures of Tetrahymena pyriformis W incorporate exogenous 3-[14C]-cilienic acid and gamma-[1(-14)C] linolenic acid, terminal products of unsaturated fatty acid synthesis, into glycerophosphatides without randomization of the radiolabel. There was no difference in the rate of loss of each of the two acids at 15 or 28.5 degrees C. Differential turnover of these fatty acids, therefore, does not appear to be the cause of the shift in fatty acid pattern observed with temperature reduction.     

Age-dependent modifications in membrane lipids: lipid composition, fluidity and palmitoyl-CoA desaturase in Tetrahymena membranes

The membrane lipid composition of Tetrahymena pyriformis NT-I was observed to change in a manner markedly dependent on the progress of culture age. The pellicular, mitochondrial and microsomal membranes were isolated from cell harvested at various growth phases (I, early exponential; II, mid-exponential; III, late exponential; IV, early stationary; V, late stationary) and their lipid composition was analyzed by thin-layer and gas-liquid chromatography. Although the phospholipid composition varied somewhat among membrane fractions, the most general age-dependent alteration was a considerable decrease in the content of phosphatidylethanolamine accompanied by a small increase in phosphatidylcholine. The 2-aminoethylphosphonolipid, enriched in the surface membrane pellicle, did not undergo a consistent change. As for fatty acid composition the most notable variation occurred in unsaturated fatty acids; a great increase in oleic and linoleic acids and a compensatory decrease in palmitoleic acid. This resulted in an augmented unsaturation of the overall phospholipid fatty acid profile of the aged membranes. The age-associated drastic decline in the palmitoleic acid content in membrane phospholipids could be accounted for by the markedly lowered activity of palmitoyl-CoA desaturase. The microsomes from the early exponential phase cells possess a 4-fold higher activity of the desaturase as compared to that of the late stationary phase microsomes. The decreased desaturase activity associated with the culture age was also reflected in the corresponding decrease in the conversion rate of [14C]palmitate to [14C]palmitoleate in cells labelled in vivo. The ESR spectra of the spin-labeled phospholipids extracted from the pellicular and microsomal membranes have led to the suggestion that these types of membrane would become more fluid with the age of growth.     

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

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

Regulation of membrane fatty acid composition by temperature in mutants of Arabidopsis with alterations in membrane lipid composition

Background  

A wide range of cellular responses occur when plants are exposed to elevated temperature, including adjustments in the unsaturation level of membrane fatty acids. Although membrane bound desaturase enzymes mediate these adjustments, it is unknown how they are regulated to achieve these specific membrane compositions. Furthermore, the precise roles that different membrane fatty acid compositions play in photosynthesis are only beginning to be understood. To explore the regulation of the membrane composition and photosynthetic function in response to temperature, we examined the effect of temperature in a collection of mutants with altered membrane lipid fatty acid composition.     

Changes in lipid fluidity and fatty acid composition with altered culture temperature in Tetrahymena pyriformis-NT1

Cultures of T. pyriformis-NT1 were grown at 20 degrees C (Tg 20 degrees C) and 38 degrees C (Tg 38 degrees C). G.L.C. analysis and D.P.H. fluorescence polarization measurements in extracted phospholipids indicated that there was increased saturation of fatty acids and relatively reduced fluidity as growth temperature was increased. Breakpoints occurred in the Arrhenius plots of fluorescence polarization at 16 degrees C for Tg 38 degrees C total extracted phospholipids and 9 degrees C for Tg 20 degrees C lipids.     

Population growth kinetics and bulk membrane lipid alterations in Tetrahymena pyriformis: exposure to pentachlorophenol

This study describes effects of exposure of the freshwater ciliate Tetrahymena pyriformis to the "classic" weak acid respiratory uncoupler pentachlorophenol (PCP) on the population growth kinetics and membrane lipid profiles. The assessment of growth kinetics of naive populations exposed to PCP, at concentrations eliciting <50% growth inhibition, showed generation times of exposed cultures similar to generation times of controls but preceded by a short lag phase (<2 h). Assessment of exposed cultures exhibiting >50% growth inhibition revealed generation times that increased with increasing concentrations of toxicant. In addition, the relative percentages of selected fatty acid methyl esters (FAMEs) in both pellicle and mitochondrial membranes were examined. Upon exposure to PCP the relative percentages of FAMEs 12:0, 14:0, 16:0, 16:1, and 18:0 did not change. However, with exposure to PCP a decrease was observed for FAMEs 15:0 and 17:0. Conversely, with PCP exposure there was an increase in FAME 18:1. A comparison of these results with those elicited upon exposure to the model narcotic 1-octanol reveals marked differences in both growth kinetics and fatty acid shifts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differences in membrane fluidity and fatty acid composition between phenotypic variants of Streptococcus pneumoniae

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P < or = 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P < or = 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P < or = 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition.     

The regulation of peroxisomal enzyme systems of Tetrahymena pyriformis by fatty acid composition, glucose and oxygen in the medium

The effect of the chain length of fatty acids on peroxisomal enzyme activities of Tetrahymena pyriformis was investigated. The growth of cells and the activities of peroxisomal enzymes were inhibited markedly by the addition of medium-chain fatty acids (C6-C12) to the culture medium, whereas the addition of longer-chain fatty acids (C14-C18) resulted in a slight increase of growth and in the marked stimulation of enzyme activities concerned with fatty acid beta-oxidation and the glyoxylate cycle in peroxisomes. Peroxisomal beta-oxidation (fatty acyl-CoA oxidase) was more potent towards longer-chain fatty acids than the mitochondrial activity (fatty acyl-CoA dehydrogenase). The induction of the peroxisomal beta-oxidation system by palmitate was repressed both by the addition of glucose and the aeration of the culture medium, whereas that of the peroxisomal glyoxylate cycle was repressed only by the addition of glucose to the medium. These results indicate that peroxisomal enzyme systems related to the beta-oxidation of fatty acids and the glyoxylate cycle are regulated by the compositions of fatty acids, glucose, and oxygen in the medium.     

Temperature-dependent changes in phospholipid and fatty acid composition and membrane lipid fluidity of Yersinia enterocolitica

Temperature-dependent compositional changes of phospholipids and their fatty acids were analysed in Yersinia enterocolitica grown at 5°, 25° and 37°C. The relative amounts of the four phospholipids, phosphatidylethanolamine (75–78%), phosphatidylglycerol (10–11%), cardiolipin (<7%) and lysophosphatidylethanolamine (<5%), were essentially the same at all growth temperatures. The degree of fatty acid unsaturation of the four phospholipids increased with decrease in growth temperature, mainly due to an increase of C_16:1 and C_18:1 and a corresponding decrease of C_16;0, C_18:0 and cyclo C_17:0. An electron spin resonance spectroscopic study of the membrane lipids showed that membrane lipid fluidity was enhanced by decreasing the growth temperatures. The changes in fatty acid composition of phospholipids in response to varied temperatures were consistent with the temperature-dependent changes in the membrane lipid fluidity of Y. enterocolitica , and were similar to those reported for other bacteria.