Kinetic changes of the erythrocyte (Mg2+ + Ca2+)-adenosine triphosphatase of rats fed different fat-supplemented diets.

The activation by Mg2+, in the presence of 0.2 mM Ca2+, of the erythrocyte ATPase from rats fed with six different fat-supplemented diets has been studied. A sigmoid kinetic curve was found. The values of the Hill coefficient showed a positive correlation with the membrane fatty acid fluidity, which is expressed as the ratio between double bond index and saturated fatty acid content. The values of the Hill coefficient ranged from 1.0, in animals fed with lard-supplemented diet, to 2.0, in animals fed with corn oil-supplemented diet. When the effect of increasing Ca2+ concentration in these two groups was studied at pH 8.1, an activation with the latter group and an inhibition with the former one were found. The activation by Ca2+ found in corn oil-fed animals was lost after treatment with phospholipase C and restored after the addition of homologous phospholipids. The activation could not be restored by addition of phospholipids from lard-fed animals. In this group, treatment with phospholipase C left the kinetic behavior unmodified, but an activation by Ca2+ could be detected after adding phospholipids from corn oil-fed animals. It is suggested that membrane fatty acid fluidity is involved in the cooperative transitions and cryptic activity of the (Mg2+ + Ca2+)-ATPase.     

Membrane lipid fatty acids and regulation of membrane-bound enzymes. Allosteric behaviour of erythrocyte Mg2+-ATPase, (Na+ + K2+)-ATPase and acetylcholineasterase from rats fed different fat-supplemented diets

Studies were carried out to determine the Hill coefficients for the inhibition by F^? of the erythrocyte membrane-bound Mg²⁺-ATPase, $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase from rats fed with seven different diets. Five groups were fed with different natural fats or oil supplements, one with a hydrogenated fat supplement and the other with fat-free diet. The responses of the red cell fatty acids to dietary fats were recorded. The value of $\text{n}$ for the inhibition by F^? of the three enzymes revealed a particular and different behaviour in each group. Correlations between the fatty acid compositions of erythrocyte membranes and cooperativity of each enzyme were calculated. The results indicate that neither the essential fatty acid family nor the non-essential ones are particularly involved in the allosteric phenomena. The increase of the double bond index/saturation ratio of fatty acids, which is taken as indicative of membrane fluidity, was accompanied in an inverse manner by changes in allosteric transitions of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase, whereas the Mg²⁺-ATPase was not dependent on this ratio. Diminution of membrane fluidity, carried out by in vitro increase of its cholesterol content, yields confirmatory results of this regulatory mechanism since the value of $\text{n}$ for acetylcholinesterase shifted as predicted.These facts indicate that the membrane fluidity is a physiological regulator for the allosteric behaviour of the membrane-bound enzymes and that each enzyme exhibits a particular behaviour in this phenomenon.     

Effect of fatty acid deficiency on microsomal membrane fluidity and cooperativity of the UDP-glucuronyltransferase

The effect of fat deprivation on microsomal membrane fluidity of guinea-pig livers and the kinetic cooperativity of UDP-glucuronyl transferase towards its natural substrate, the UDP-glucuronic acid, were studied. Fat deprivation in the diet of weanling guinea-pigs evoked a typical essential fatty-acid-deficient pattern in the composition of the microsomal membrane. The unsaturated:saturated fatty acid ratio progressively declined in the membrane during the 21-day period tested. This decline determined a gradual increase in the fluorescence anisotropy (rs) of the membrane labeled with diphenylhexatriene and the apparent microviscosity of the lipid bilayer calculated from these values increased from 1.1 to 1.8 poise. In addition, when the infinitely slow decaying fluorescence anisotropy ((r infinity), which is proportional to the square of the lipid order parameter, was calculated from rs data, a significant increase in these parameters was also obtained. Furthermore, this decrease of the double bond index:saturated acid ratio of the membrane was associated with a parallel increase in Hill coefficients of the UDP-glucuronyl transferase that gradually lost the negative homotropic effect and cooperativity of UDP-glucuronic acid. The Hill coefficient varied from 0.39 to 0.98 during the 21-day period studied. Our observations indicate on one side that changes in the fat composition of the diet are accompanied by modifications in the lipid composition and fluidity of the microsomal membrane, and the apparent cooperativity of the enzyme. On the other side, they suggest that the evaluation of Hill coefficients of UDP-glucuronyl transferase might be used as a sensitive test to investigate conformational changes in the microsomal membrane of the liver.     

Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration.

A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5'-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5'-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration.     

Growth characteristics and polyene sensitivity of a fatty acid auxotroph of Candida albicans.

A fatty acid auxotroph of Candida albicans 6406, designated A' 44 and originally isolated as an oleic acid requiring strain, has been shown to be a delta9 desaturase mutant. Although lacking this step in fatty acid biosynthesis, it appears to retain the ability to desaturate monounsaturated fatty acids. The polyene sensitivity of the organism grown on different fatty acid supplements varied between 0-08 +/- 0-02 and 1-20 +/- 0-30 microgram amphotericin B methyl ester ml-1 for exponentially growing cells. In spite of this variation, the sterol composition remained fairly constant, the major differences lying in fatty acid composition. Stationary-phase cells were more resistant to amphotericin B methyl ester, although again this change was not associated with changes in sterol content. The organism was most resistant when grown in the presence of oleic or linoleic acid. Protoplasts derived from resistant organisms grown on these two fatty acids were also resistant, indicating that the structure of the cell wall was less important than that of the plasma membrane in determining polyene sensitivity under these conditions.     

Kinetic properties of soluble and membrane-bound acetylcholinesterase from electric eel

Using electric eel acetylcholinesterase (AChE) which was either membrane-bound (AChEm) or solubilized (AChEs), similar kinetics were seen in the absence of inhibitor or in the presence of edrophonium, trimethylammonium ion or paraoxon. Thus, both forms of the enzyme appear to behave similarly toward various inhibitors. However, in the presence of a probe sensitive to allosteric effects or changes in membrane fluidity, the two forms exhibit altered behavior. In the presence of F-, the relative rate of substrate hydrolysis by AChEm was reduced more rapidly than with AChEs, whether or not paraoxon was present. When inhibition by paraoxon (10(-7)-10(-4) M) was studied in the presence of F-, AChEs had a Hill coefficient of 1.0, whereas with AChEm the Hill coefficient changed from 0.8 to 1.5.     

Biogenesis of microbial transport systems: evidnce for coupled incorporation of newly synthesized lipids and proteins into membrane

The biogenesis of the independent β-galactoside and β-glueoside transport systems of Escherichia coli K12 has been studied using an unsaturated fatty acid auxotroph. The response of transport rate to temperature was determined for cells grown with different fatty acid supplements. A change in the slope of an Arrhenius plot for transport rate was obtained at transition temperatures unique for each of the five fatty acid supplements tested. Both of the transport systems studied here had identical transition temperatures when the cells were grown with the same fatty acid supplement, indicating that the transport temperature characteristics are determined primarily by the properties of the lipid phase of the membrane.     

Involvement of membrane lipids in radiation damage to potassium ion permeability of Escherichia coli.

Radiation damage to K+ permeability of an unsaturated fatty acid auxotroph of E. coli grown with oleate or linolenate was investigated at different temperatures. A remarkable effect of radiation was observed at 0 degrees C with cells that had been grown with linolenate at 42 degrees C. This indicates that, besides protein, membrane lipids at least are involved in the radiation damage. The damage also seems to be affected by the fluidity of membrane lipids.     

Maturation of bacteriophage 9NA DNA is influenced by the fatty acid composition of the host cell membrane

The fatty acid composition of the membrane of the conditional auxotroph fabB2 can be altered by allowing the cells to grow at non-permissive temperature (37°C) in the presence of a cis-unsaturated fatty acid. The phage 9NA, a virulent phage ofSalmonella typhimurium, can not multiply in fabB2. Synthesis and maturation of the phage DNA are differentially affected by variation in the fatty acid composition of the cell membrane. The replicating DNA associates with the membrane complex, the site of DNA synthesis. The association is comparatively weak in oleic, claidic, palmitoleic, palmitelaidic and linolelaidic acid enriched cells. When the cells are grown in the presence of palmitoleic acid, a large pool of concatemeric phage DNA accumulates in the cytoplasm within 10 min of infection. The conversion of concatemeric DNA to monomeric one i.e., mature phage length DNA, is inhibited in such cells. The presence of concatemeric DNA can be visualized by electron microscope. Such a situation is not observed when the cells are grown in media supplemented with other types of unsaturated fatty acids. The mechanism by which the host cell membrane lipid controls phage development is yet to be worked out.     

Effects of Fatty Acid Substitution on the Release of Enzymes by Osmotic Shock

The release of enzymes by osmotic shock from Escherichia coli strain 30E, an unsaturated fatty acid auxotroph, was examined in culture supplemented with either cis- or trans-unsaturated fatty acids. Cultures grown in oleate-supplemented medium release a large fraction of the total cyclic phosphodiesterase, acid hexose phosphatase, and 5'-nucleotidase following osmotic shock. Cultures grown in elaidate-supplemented medium release much less of these same enzymes after shock treatment. Cultures grown with either supplementation show total release of these enzymes upon conversion to spheroplasts, demonstrating that the enzymes are in the periplasmic space in both cases. Cultures grown with either oleate or elaidate as fatty acid source were washed and suspended in medium containing the other isomer. The change from oleate to elaidate resulted in a rapid decrease in ability of the cells to release the three enzymes after osmotic shock so that within a 25% increase in cell mass the culture responded to osmotic shock as would a culture grown overnight in elaidate-supplemented medium. The reverse experiment resulted in a gradual increase in the ability of the cells to respond to osmotic shock. The outer membrane of E. coli is altered by the incorporation of elaidate, as indicated by electron microscopic data.     

Influence of the incorporation of cholesterol on the doubling time and on the arrhenius and hill plots of two membrane-bound enzymes ofEscherichia coli K12

Escherichia coli was grown at 37°C with increasing cholesterol concentrations in the growth medium. Under these conditions, it was shown that neither the phospholipid species nor the fatty acid composition were affected by the incorporation of cholesterol. On the other hand, the doubling time (t_d) was increased; two membrane-bound enzymes, (Ca⁺⁺)-ATPase and D-lactate-dehydrogenase, were modified in the temperature-dependence activities, and the (Ca^±±)-ATPase changed its Hill coefficient for the inhibition by Na⁺. This last parameter was the most sensitive in detecting very low cholesterol incorporations to the cytoplasmic membranes.     

Relationship between Chloroplast Membrane Fatty Acid Composition and Photosynthetic Response to a Chilling Temperature in Four Alfalfa Cultivars

The photosynthetic responses of four alfalfa (Medicago sativa L.) cultivars to 10 and 22 C air temperatures were examined and the relationship between the photosynthetic response at 10 C and the fatty acid composition of the chloroplast membranes was determined. Chilling-resistant cultivars exhibited moderate reductions in photosynthesis at 10 C, compared to 22 C, and contained a significantly greater percentage of polyunsaturated fatty acids in the chloroplast membrane and a greater double bond index than the chilling-sensitive cultivars. The chilling-sensitive cultivars exhibited severe reductions in photosynthesis at 10 C, compared to 22 C. The reduction in photosynthesis at 10 C is shown to be negatively correlated (r = −0.94) with the double bond index of the chloroplast membranes of the cultivars observed.

The results support the hypothesis that reduced photosynthesis due to chilling temperatures is influenced by the unsaturated fatty acid composition of the chloroplast membrane which affect temperature-induced phase changes in chloroplast membrane lipids.

     

Gastric lipolysis in the developing rat. Ontogeny of the lipases active in the stomach

Two nutritional models, an essential fatty acid deficiency model and the feeding of saturated versus unsaturated fats, were used in a feeding study in order to assess the relationship between tissue fatty acid composition and the activities of some membrane-associated enzymes. Purified diets containing 7% hydrogenated coconut oil, 7% corn oil, 10% safflower oil or butter were fed to rats for a total of 49 weeks (1 week of pregnancy, 3 weeks of lactation and 45 weeks post-weaning). Tissue homogenates from submandibular salivary glands and kidneys were analyzed for fatty acid composition of total lipids and phospholipids. Changes in fatty acid patterns typical of essential fatty acid deficiency such as an increase in the levels of 16:1 and 18:1, a decrease in 18:2 and 20:4 and an accumulation of 20:3 omega 9 were observed in salivary glands and kidneys of rats fed the deficient diet. Tissues of rats fed 10% butter also showed fatty acid compositional changes which were somewhat similar to those in essential fatty acid deficiency, but to a lesser degree. The activities of ouabain-sensitive (Na+ + K+)-ATPase were higher in homogenates of salivary glands and kidneys of the deficient rats and those fed butter as compared with their controls. The results suggest a relationship between the double bond index of fatty acids as an indication of membrane lipid fluidity and allosteric modification of (Na+ + K+)-ATPase activity. However, other explanations for the observed changes in (Na+ + K+)-ATPase activity cannot be ruled out. There were no diet-related differences in the activities of gamma-glutamyltranspeptidase or 5'-nucleotidase.     

Lipid profiles of conidia of Aspergillus niger and a fatty acid auxotroph

Conidial lipids of the wild-type (V35) Aspergillus niger and its unsaturated fatty acid auxotroph (UFA2) were compared. The wild type contained lower levels (7.6%) of phospholipids and higher levels (28.4%) of glycolipids than the mutant (16.5 and 22.2%, respectively). Oleic (33.4%), linoleic (22.5%), palmitic (12.8%), stearic (7.4%), and linolenic (6.2%) were the main fatty acids of the wild type (V35). The mutant grew only in the presence of unsaturated fatty acid having at least one delta 9cis double bond, and its conidial fatty acid profile was influenced by the exogenous acid. Analyses of the fatty acids of UFA2 grown in the presence of different fatty acid supplements support the original view that the mutant is defective in delta 9-desaturase activity.     

LM cell growth and membrane lipid adaptation to sterol structure

Using a sterol auxotroph of the LM cell mouse fibroblast, we demonstrate that relatively few cholesterol analogues can substitute for cholesterol as a growth factor. The auxotroph grows normally on desmosterol and trans-22-dehydrocholesterol and at reduced rates on dihydrocholesterol, campesterol, and 22,23-dihydrobrassicasterol. It does not grow with beta-sitosterol, stigmasterol, ergosterol, or cis-22-dehydrocholesterol when the sterol is present as sole supplement but does grow at normal rates when the analogue is supplied with suboptimal amounts of cholesterol. Two contrasting types of membrane lipid changes are observed in cells grown on cholesterol analogues. In cells grown with dihydrocholesterol, a marked increase in desaturation and elongation of fatty acids is noted. Conversely, when cells are grown with cis-22-dehydrocholesterol, desaturation and elongation of fatty acids are severely curtailed. Cells grown on alkyl sterols respond like cells grown on cis-22-dehydrocholesterol but in a less pronounced fashion. The effects of sterol substitution in mammalian cells versus in lower eukaryotes are compared, and an explanation for the secondary changes in fatty acid composition in terms of phospholipid phase behavior is suggested.     

Membrane cooperative enzymes. High molecular specificity for blocking action of thyroxine on triiodothyronine effect in rat erythrocyte and Escherichia coli systems.

The molecular specificity for the blocking action of thyroxine on the triiodothyronine effect in the cooperativity of membrane-bound rat erythrocyte acetylcholinesterase and Escherichia coli Ca2+-ATPase was analyzed. Changes in the values of n (Hill coefficient) were obtained at strict physiological levels of these hormones. The structural requirements of the thyroid hormones to modify the membrane-bound systems were studied using various analogues of these hormones. In the erythrocyte system, a very high molecular specificity for triiodothyronine and thyroxine actions was found. The L-alanine side is essential to carry out both the allosteric desensitization and the blocking effects. The blocking ability of thyroxine is characterized by the presence of iodine in the 5' position. The bacterial system presented only specificity for the triiodothyronine allosteric desensitization. A system of membrane-bound enzymes for the study of the actions of thyroid hormones, is presented here.     

Influence of ethanol on fatty acid composition of phospholipids in cultured neurons

Animals chronically exposed to ethanol show changes in neural membrane lipids which may underlie the development of tolerance and physical dependence. The object of this study was to investigate changes in the fatty acid composition of neuronal phospholipids cultured in the presence of ethanol (55 or 110 mM) for periods up to 7 days. Decreases were observed in the percentage of individual and total saturated fatty acids, while the double bond index: total saturated fatty acid ratio, increased. These changes do not support the hypothesis that neural membrane lipid composition changes to counteract the fluidizing action of ethanol.     

Effect of supplementation with exogenous fatty acid on the biological properties of a fatty acid requiring auxotroph ofSalmonella typhimurium

The effects of changes in fatty acid composition of the cell membrane on different biological functions ofSalmonella typhimurium have been studied with the help of a temperature sensitive fatty acid auxotroph which cannot synthesise unsaturated fatty acids at high temperature. On being shifted to nonpermissive temperature the cells continue growing for another one and half to two generations. The rates of protein and DNA syntheses run parallel to the growth rate but the rate of RNA synthesis is reduced. Further, there is a gradual reduction in the rate of transport of exogenous uridine and thymidine into the soluble pool. The transport process can be restored by supplementing the growth medium with cis-unsaturated fatty acids but not trans-unsaturated ones although the growth of the cells is resumed by supplementation with eithercis or trans-unsaturated fatty acids. However, supplementation withtrans, trans-unsaturated fatty acids leads to only partial recovery of the transport process. The rate of oxygen uptake is also affected in cells grown in the presence of thetrans-unsaturated fatty acids, elaidic acid and palmitelaidic acid. Analysis of cells grown under different fatty acid supplementation indicate that fatty acid composition of the cell membrane, especially the ratio of unsaturated to saturated fatty acids varies with temperature shift and supplementation of the growth media with fatty acids.     

Phospholipid fatty acid composition affects enzymatic antioxidant defenses in cultured Swiss 3T3 fibroblasts

Summary

The aim of this work was to study the adaptation of enzymatic antioxidant cell defense to the nature of the membrane polyunsaturated fatty acids (PUFA). 3T3 Swiss fibroblasts were grown for 5 days in a medium supplemented with 50 μM linoleic acid (LA) or eicosapentaenoic acid (EPA) and compared t control cells (C). The phospholipid fatty acid content was evaluated: LA were enriched in n-6 PUFA (27.8%) in comparison to C (6.7%) or EPA (5.6%); EPA were enriched in n-3 PUFA (26.2%) in comparison to LA (4.4%) or C (4.6%). The fatty acid double bond index (DBI) increased from C to LA and EPA. The activities of the three key enzymatic antioxidant defenses, SOD, GPx and GST, increased with the degree of unsaturation of the phospholipid fatty acids. In the cells with fatty acids that are very sensitive to oxidative stress, the higher activities of SOD and GPx might act to limit the initiation of lipid peroxidation and the higher activities of GST and GPx to decrease the toxic effects of the various species produced from lipid degradation.