Membrane Unsaturated Fatty Acids Research Articles

Electron spin resonance studies of lipid fluidity changes in membranes of an uncoupler-resistant mutant of Escherichia coli

The fluidity of the lipids in membrane preparations from a mutant of Escherichia coli resistant to the uncoupler CCCP, grown at different temperatures with and without CCCP, was examined by electron spin resonance using the spin probe 5-doxyl stearic acid. The fluidity of the membrane lipids at the growth temperature, as estimated using electron spin resonance, was less in cells grown at lower temperatures. Precise homeoviscous adaptation was not observed. Growth in the presence of CCCP resulted in a decrease in membrane lipid fluidity, particularly in the inner (cytoplasmic) membrane. There was no change in the proportion of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in the cell envelope. However, there was an increase in the proportion of unsaturated fatty acids in membranes from cells grown with uncoupler. This was reflected in the increased fluidity of the lipids extracted from these membranes. This result is contrary to that expected from measurements of the fluidity of the lipids extracted from these membranes. This result is contrary to that expected from measurements of the fluidity of the lipid in these membranes. The decreased fluidity of the lipid in these membranes may be a consequence of the observed increase in the ratio of protein to phospholipid.

Dietary linoleic acid modulates liver plasma membrane unsaturated fatty acid composition, phosphatidylcholine and cholesterol content, as well as glucagon stimulated adenylate cyclase activity

Male Sprague-Dawley rats were fed diets containing 20% (w/w) fat in which linoleic acid was substituted for saturated fatty acids. High or low levels of dietary linoleic acid were fed for 24 days. Liver plasma membranes were isolated for analysis of lipid composition and glucagon stimulated adenylate cyclase activity. Dietary saturated fatty acid content did not affect the saturated fatty acid composition of membrane phospholipids. Increase in dietary linoleic acid increased content of linoleic acid homologues in phosphatidylcholine. Diets low in linoleic acid increased total phosphatidylcholine and cholesterol levels in the plasma membrane, increasing glucagon stimulated and fluoride stimulated adenylate cyclase activity. These observations imply that change in glucagon stimulated adenylate cyclase activity resulted from transitions in membrane phospholipid content of linoleic acid and its homologous fatty acids or from associated changes in membrane phospholipid class content.

Does an increase in membrane unsaturated fatty acids account for Tween 80 stimulation of glucosyltransferase secretion by Streptococcus salivarius?

When Streptococcus salivarius was grown in batch culture in the presence of various Tween detergents, the fatty acid moiety of the detergent was incorporated into the lipids of its membrane. Tween 80 (containing primarily oleic acid) markedly stimulated the production of extracellular glucosyltransferase and also increased the degree of unsaturation of the membrane lipid fatty acids. The possibility that an increase in membrane unsaturated fatty acids promoted extracellular glucosyltransferase production was examined by growing cells at different temperatures in the presence or absence of Tween 80. The membrane lipids of cells grown at 30 degrees C, 37 degrees C and 40 degrees C without Tween 80 exhibited unsaturated/saturated fatty acid ratios of 2.06, 1.01 and 0.87 respectively. A significant increase in the production of extracellular glucosyltransferase was observed at 30 degrees C compared to cells grown at 40 degrees C. However, cells produced much more exoenzyme at all temperatures when grown with Tween 80. The results indicated that an increase in the unsaturated fatty acid content of the membrane lipids was not by itself sufficient to account for the stimulation of extracellular glucosyltransferase production by Tween 80, but that the surfactant also had to be present.

Toxicity of activated oxygen: Lack of dependence on membrane unsaturated fatty acid composition

Membrane unsaturated fatty acid oxidation has been suggested as a mechanism of toxicity for a variety of activated oxygen species. We have tested this hypothesis by manipulating the fatty acid composition of an Escherichia coli mutant that is unable to synthesize unsaturated fatty acids. To provide a wide range of susceptibility to membrane oxidation we have replaced the naturally occurring monoenoic acyl chains with cyclopropanes to greatly reduce the unsaturation level and with linoleate to increase the membrane unsaturation. These cultures were treated with ozone, hydrogen peroxide, singlet oxygen and paraquat. In no case was there substantial protection from toxicity afforded by cyclopropanes nor was there enhancement of toxicity to cells with the polyunsaturated membranes. We suggest, therefore, that oxidation of membrane unsaturated fatty acids is not an essential component of the toxicity to E. coli of active oxygen species.

Requirements of Δ9 and Δ12 fatty acid desaturation in Neurospora

Microsomes prepared from the wild-type strain and lipid auxotrophs of Neurospora were analyzed for Δ 9-(stearoyl- CoA) and Δ 12-(oleoyl- CoA) desaturase activities. The wild-type Δ 9-desaturase was found to have a 20-fold higher specific activity and 2-fold lower activation energy than the Δ 12-desaturase. In addition, Δ 12-desaturase had higher K m app values for oleoyl-CoA and for NADH than the equivalent values for Δ 9-desaturase. These properties were correlated with a rate-limiting role of Δ 12-desaturase in the production of 18:2, the major fatty acid of Neurospora. The Δ 12-desaturase also exhibited a higher tolerance to pH changes and to cyanide than did the Δ 9-desaturase. Both activities could be measured in the same reaction mixture using stearoyl-CoA as the substrate, indicating a coupling of the two enzymes. Enrichment of cellular membranes of the wild-type Neurospora with 18:0 and 18:1, 18:2, 18:3 fatty acids led to the conclusion that the presence of excess substrate in the membrane induces activation of the appropriate desaturase. These experiments also suggested that the membrane fluidity, as determined by the degree of unsaturation of membrane fatty acids, may influence the activities of the desaturating enzymes. Perturbation of the polar head groups of the membrane phospholipids indicated that the correct composition of anionic phospholipids is an absolute requirement for the function of both desaturases. These studies show that the activities of the Δ 9-desaturase and the Δ 12-desaturase are regulated by a variety of factors and that the Δ 12-desaturase is subjected to less stringent controls than the Δ 9-desaturase.

Low temperature acclimation of root fatty acid composition, leaf water potential, gas exchange and growth of soybean seedlings

Abstract Root fatty acid composition, photosynthesis, leaf water potentials, stomatal resistances, leaf specific weights, and root: shoot ratios of soybean were measured in two temperature regimes.Groups of soybean plants were grown in controlled chambers of the Duke University Phytotron under two thermoperiods. One group of the plants was grown from seed for 3 weeks in either 29/23°C or 17/11°C thermoperiods, and another group was grown for 2 weeks in 29/23°C and then transferred to the 17/11°C thermoperiod where it remained for 8 days. Broccoli was also grown in either 29/23°C or 17/11°C thermoperiods.Soybean roots contained more unsaturated fatty acids than broccoli roots, although broccoli roots showed a larger increase in unsaturation than soybean roots with decreased temperature. The fatty acid unsaturation in the roots of soybean began to increase rapidly after the temperature regime was changed. The increase was in the new roots produced in the cold regime rather than in the pre‐existing roots.The soybean leaf water and osmotic potentials decreased about 0.4 MPa, beginning one day after the transfer from 29/23°C to 17/11°C, but recovered significantly after 8 d.Plants grown at 17/11 °C had lower rates of photosynthesis and adaxial stomatal resistances, but higher root: shoot ratios and specific leaf weights compared to plants grown at 29/23°C. Plants grown and maintained at 29/23°C showed a steady increase in photosynthetic rates over the 8‐d experimental period, whether rates were measured in 1 mol m−3 or 9 mol m−3 oxygen. Plants transferred to 17/11°C however maintained constant rates of photosynthesis at 1 mol m−3 O2, whereas at 9 mol m−3 rates declined for 2 d then were constant for the remaining 6 d of the experimental period.These results suggest that changes in membrane fatty acid unsaturation is an important aspect of plant acclimation to chilling temperatures in terms of maintaining root permeability and water uptake. However, the degree of unsaturation is not a good indicator of differences in chilling tolerance among species. The apparent acclimation of photorespiration to a constant percentage of photosynthesis suggests a role of photorespiration in the plant.

Copper-mediated Lipid Peroxidation Processes in Photosynthetic Membranes

The phytotoxic effect of Cu via the photosynthetic electron transport system was studied with isolated spinach chloroplasts. Cu(II) ions induce a light-driven peroxidation of membrane lipids leading to ethylene formation, the latter dominating over a concurrent ethane production. Seemingly, the hydroxyl radical originating from superoxide anion is the starting reactive O(2) species. Cu ions inhibit photosynthetic electron transport and apparently catalyze the formation of hydroxyl radical and Fenton-type reactions that result in destruction of unsaturated membrane fatty acids. The concept on the mode of action of Cu(II) and Cu(I) ions in lipid peroxidation as presented here suggests the influence of Cu on different reactions. Two sites are in the photosynthetic redox system; Cu participates in two Fenton-type reactions and in the conversion of ethyl radical to ethylene and ethane.

Role de l'oxygene et de la temperature sur la composition en acides gras des cellules isolees d'Erable ( Acer pseudoplatanus L.)

Temperature and oxygen effects on the degree of unsaturation of membrane fatty acids have been investigated with sycamore cells in suspension culture. Sycamore cells were incubated with [ 14C] acetate at temperature varying from 15 to 25°C and at O 2 concentration from 12.5 to 305 μM. It was found that: 1. (i) no significant difference was observed in the distribution of radioactivity between oleate and linoleate with different temperatures; 2. (ii) in marked contrast, the aeration conditions during growth of plant cell cultures affected the fatty acid pattern of the total lipids: by maintaining the oxygen concentration below 60 μM, the molar proportion of oleate increased dramatically whereas that of the linoleate decreased. Under these conditions, the aeration of the culture medium (250 μM) induced a rapid transformation of oleate to linoleate. These results cast further doubt on the importance of the temperature on the degree of unsaturation of the fatty acids in sycamore cells, but confirmed evidence that the formation of unsurated fatty acids by plant cells was indeed controlled by the oxygen concentration in solution.

Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells

The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI +D +, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI +D −, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI +D − compared to the MGI +D + clones. The MGI +D − clones produced an unusual polyunsaturated C 20:5 fatty acid at 28°C, whereas the MGI +D + clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI +D − clones than of the MGI +D + clones. The cells of MGI +D + clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.

Cold sensitivity of heart rate and succinate oxidation by myocardial homogenates from hibernating ground squirrels and diet-treated rats

Feeding rats a highly unsaturated fat diet, when compared with those fed a highly saturated fat diet, lowered the temperature at which discontinuity in the kinetics of succinate oxidation by myocardial homogenates occurred, raised the apparent energy of activation both above and below the breakpoint temperature, and decreased the mean temperature below which heart rate became irregular. With respect to these criteria, the rats fed unsaturated fat showed characteristics similar to those of hibernating ground squirrels. However, preliminary myocardial phospholipid fatty acid analyses, heart rate-body temperature relationsihps, and the ability to survive profound hypothermia were not appreciably changed by dietary treatment. We conclude that manipulation of dietary fat composition, at least under the circumstances tested, has only a limited ability to influence cold tolerance, that the kinetics of membrane-bound enzymes may not be valid indicators of whole animal cold tolerance and that the degree of membrane fatty acid unsaturation may not determine cold tolerance of mammals.

Spectrofluorescent detection of in vivo red cell lipid peroxidation in patients treated with diaminodiphenylsulfone.

In the absence of vitamin E deficiency, red cell lipid peroxidation has not been clearly demonstrated in freshly drawn blood obtained from patients with various hemolytic anemias despite indirect evidence that oxidative decomposition of cell membrane unsaturated fatty acids occurs in these particular hemolytic states. Recent studies have indicated that malonaldehyde, a decomposition product of oxidized polyunsaturated fatty acids, is able to covalently cross-link the amino groups of protein or lipid resulting in a fluorescent compound. In the present study we have utilized spectrofluorescent technique to assess whether such fluorescence is present in red cell lipid extracts in association with lipid peroxidation. In vitro red cell lipid peroxidation produced by ultraviolet radiation or the oxidant gas ozone was associated with the development of a fluorescent peak (excitation maximum 360 nm; emission maximum 440 nm) in lipid-containing red cell extracts Similar fluorescence was observed after incubation of red cells with malonaldehyde or with malonaldehyde-containing extracts of peroxidized red cell lipid. Spectrofluorescent evaluation of chloroform: isopropanol extracts obtained from the freshly drawn red cells of six patients receiving the oxidant hemolytic drug diaminodiphenylsulfone also revealed a peak at 440 nm which ranged from 39 to 78 U. In contrast, the levels in samples obtained from 11 hematologically normal subjects were 17-27 fluorescence U. No evidence for an increase in blood levels of free malomaldehyde was observed using the 2-thiobarbituric acid test which is the most commonly performed assay of lipid peroxidation. Serum vitamin E levels were within the normal range. Density separation indicated that the bulk of the fluorescence was present in older red cells. A similar fluorescent peak was also observed in lipid-containing extracts of red cells obtained from rabbits repeatedly injected with phenylhydrazine. The finding of fluorescent spectra consistent with the cross-linking of aminolid by malonaldehyde in the red cells of patients receiving diaminodiphenylsulfone indicates that in vivo red cell lipid peroxidation does occur in the absence of vitamin E deficiency.

Environmental effects on tetrahymena membranes : Temperature-induced changes in membrane fatty acid unsaturation are independent of the molecular oxygen concentration

The study was designed to show whether the increase in unsaturated fatty acids in Tetrahymena pyriformis membrane phospholipids with decreasing growth temperature is due to the sharp rise in the concentration of dissolved O 2 found at lower temperatures. Increasing cell density in cultures growing at a constant temperature was found to have a more drastic effect upon the O 2 level than shifting a culture from one temperature extreme to the other. However, the pattern of phospholipid fatty acid unsaturation did not vary over a 30-fold gradual decrease in O 2 concentration measured in cultures during logarithmic growth at 39.5°C, and unsaturation in 15°C cells sampled over a 5-fold decrease in O 2 concentration was also unchanged. Cells grown at 39.5°C under a constant relatively high O 2 concentration (5–6 mg O 2/l) or low O 2 concentration (0.3 mg O 2/l) had nearly identical distributions of membrane fatty acids. Only under almost completely anaerobic conditions was it possible to measure via radioactive labeling experiments an inhibition of fatty acid desaturation. Thus the increased membrane fatty acid unsaturation induced by decreasing growth temperature is caused by some factor other than the rising levels of dissolved O 2.

Biogenesis of mitochondria: The effects of membrane unsaturated fatty acid content on the activity and assembly of the yeast mitochondrial protein-synthesizing system

The steady-state unsaturated fatty acid content of yeast mitochondria was controlled by growing cells of an unsaturated fatty acid auxotroph under derepressed conditions in glucose-limited chemostat cultures. Variations in mitochondrial unsaturated fatty acid content influenced the activity and assembly of the mitochondrial protein-synthesizing system. The temperature of discontinuity of Arrhenius plots of in vitro mitochondrial protein synthetic activity increased from below 6 ° to 24 °C as the proportion of membrane unsaturated fatty acid was lowered from 75 to 44%. Arrhenius plots of the inner mitochondrial membrane functions ATPase and NADH oxidase gave similar results. When the proportion of membrane unsaturated fatty acid was reduced below about 30%, no in vitro mitochondrial protein synthetic activity could be detected. These mitochondria lacked cytochrome aa 3, had low levels of cytochrome b, and the ATPase was insensitive to oligomycin inhibition, indicating that mitochondrial protein synthesis was also inactive in vivo. No mitochondrial ribosomal subunits could be detected in mitochondria containing 28% or less of their fatty acids unsaturated. Although mitochondrial transcription occurred, the RNA fractions isolated were all of low molecular weight, and intact mitochondrial ribosomal RNA could not be detected.

Biogenesis of mitochondria: The effects of altered steady-state membrane lipid composition on mitochondrial-energy metabolism in Saccharomyces cerevisiae

A chemostat culture technique has been developed for the growth of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. Any chosen steady-state cellular unsaturated fatty acid level between 75 and 15% of the total fatty acids could be established and maintained. In all cultures the steady-state glucose concentrations were maintained at levels below that which induces catabolite repression. The efficiency of oxidative phosphorylation as determined from the molar growth yield decreased as the cellular unsaturated fatty acid composition was lowered. The number of moles of ATP produced by oxidative phosphorylation per mole of glucose utilized was 7.2, 4.8, 0.7, and 0.4 for cells in which 75, 50, 44, and 34%, respectively, of the total fatty acids were unsaturated. The lesion in oxidative phosphorylation was a direct result of lowering the membrane unsaturated fatty acid composition as the respiratory activities and cytochrome content of cells and mitochondria were unaffected by a decrease in the cellular unsaturated fatty acid level from the wild-type value of about 75% down to about 34%. In cells which contained lipids with 22–28% unsaturated fatty acids, cyanide-sensitive respiration was absent, and the levels of all mitochondrial cytochromes were less than 10% of normal. The reduction in the levels of cytochromes aa 3 and b appeared to be a consequence of a loss of mitochondrial protein synthetic activity in such cells. The level of cytochrome c was also greatly decreased, indicating that the cellular unsaturated fatty acid composition was affecting either the synthesis in the cytoplasm of mitochondrial proteins or the assembly of these proteins in the mitochondria.

Evidence for a functional association of DNA synthesis with the membrane in mitochondria of Saccharomyces cerevisiae.

We have studied the effect of membrane fatty acid composition on replicative DNA synthetic activity in mitochondria isolated from Saccharomyces cerevisiae. Cells containing different levels of membrane unsaturated fatty acids were obtained by growth of a fatty acid desaturase mutant of Saccharomyces cerevisiae in glucose-limited chemostat cultures supplemented with various concentrations of Tween 80. Arrhenius plots of DNA synthetic activity in isolated mitochondria show a discrete discontinuity at specific temperature which are dependent on the membrane unsaturated fatty acid content of the mitochondria. This indicates a functional association of DNA replication with the mitochondrial membrane in Saccharomyces cerevisiae.

Electron paramagnetic resonance spectroscy. Free radical signals associated with ozonization of linoleic acid.

The air pollutant ozone Is believed to exert its deleterious biological effects by the formation of free radicals. However these free radicals have not been directly measured. It has also been suggested that peroxidation of cell membrane unsaturated fatty acids is an important mechanism In ozone toxicity. Utilizing electron paramagnetic resonance technique, direct ozonization of linoleic acid produced measurable free radicals after a two-hour induction period.