Influence of dietary lipids on microsomal membranes from chick breast muscle

1. The effect of different dietary fat intake on the lipid composition and fluidity of microsomal membranes as well as in the enzymatic activity of the Ca2+-ATPase from chick breast muscle was investigated. 2. When a standard diet was supplemented with 10% sunflower seed oil, an increase in the relative amounts of unsaturated fatty acids and membrane fluidity and a decrease in the cholesterol content was observed. 3. The presence of 6% cholesterol in the diet does not modify the fatty acid composition and the fluidity of the membrane but increased, in a low extension, the cholesterol content. 4. The provision of the sunflower seed oil-rich diet supplemented with cholesterol just 48 hr before death promoted an increase in the relative amounts of unsaturated fatty acids and cholesterol content whereas the membrane fluidity decreased in a significant extent. 5. Despite that dietary lipids gave rise in some cases to changes in lipid composition and in the physical state of the microsomal membrane, neither the Ca2+ uptake capacity nor the ATPase activity were significantly affected.     

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.     

Effects of bilayer cholesterol on human erythrocyte hexose transport protein activity in synthetic lecithin bilayers

In this study, we describe the effects of altered bilayer cholesterol content on reconstituted, protein-mediated sugar transport. The system used was the human erythrocyte sugar transporter (band 4.5) reconstituted into the bilayers of large unilamellar vesicles. Vesicle preparations were formed from synthetic lecithins whose bilayer cholesterol content ranged from 0 to 50 mol %. Transport was measured by microturbidimetric analysis over the temperature range of 0-65 degrees C while bilayer physical state was characterized by differential scanning calorimetry. Reconstituted transport activity was irreversibly lost between 62 and 65 degrees C. The Km for reconstituted transport was found to increase only slightly with increasing temperature and was not systematically affected by bilayer cholesterol content. The most striking observation of this study is that over certain critical cholesterol concentrations, as little as a 2.5% change in bilayer cholesterol can result in as much as a 100-fold change in Vmax per reconstituted protein. Our findings run counter to the view that increasing bilayer cholesterol content monotonically transforms a membrane into a state of "intermediate fluidity". Abrupt, cholesterol-induced bilayer reorganizations occurring at 15-20 and 30 mol % bilayer cholesterol are markedly reflected in altered sugar transport rates. Increasing the cholesterol content of crystalline distearoyllecithin bilayers inhibits the activity of the reconstituted transporter. It is apparent from these studies that bilayer "fluidity" is neither the sole nor a major determinant of the Indeed, we find the effect of cholesterol on transport activity is independent of its ability to fluidize membranes.     

Cholesterol in sarcoplasmic reticulum and the physiological significance of membrane fluidity.

Vesicles of sarcoplasmic reticulum from rabbit muscle can be loaded with cholesterol to at least 20 mol% with respect to endogenous sarcoplasmic-reticulum phospholipid without effect on the ATPase activity at 32 degrees C. This applies both to sarcoplasmic-reticulum vesicles in which the ATPase activity is stably coupled to Ca2+ accumulation, and to sarcoplasmic-reticulum vesicles in which the sarcoplasmic-reticulum ATPase is activated severalfold by fully uncoupling the enzyme from net Ca2+ accumulation. Since the incorporation of cholesterol causes a large decrease in fluidity of sarcoplasmic-reticulum phospholipid bilayer, these results for sarcoplasmic reticulum raise the more general question of whether bilayer fluidity is important in modulating the function of membrane proteins under physiological conditions as is widely assumed, or whether the function of membrane proteins may be effectively buffered under normal operating conditions against changes in bilayer fluidity due to extraneous agents.     

Glucagon-stimulated adenylate cyclase detects a selective perturbation of the inner half of the liver plasma-membrane bilayer achieved by the local anaesthetic prilocaine.

Prilocaine can increase the fluidity of rat liver plasma membranes, as indicated by a fatty acid spin-probe. This led to the activation of the membrane-bound fluoride-stimulated adenylate cyclase activity, but not the Lubrol-solubilized activity, suggesting that increased lipid fluidity can activate the enzyme. With increasing prilocaine concentrations above 10 mM, the membrane-bound fluoride-stimulated activity was progressively inhibited, even though bilayer fluidity continued to increase and the activity of the solubilized enzyme remained unaffected. Glucagon-stimulated adenylate cyclase was progressively inhibited by increasing prilocaine concentrations. Prilocaine (10 mM) had no effect on the lipid phase separation occurring at 28 degrees C and attributed to those lipids in the external half of the bilayer, as indicated by Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and the order parameter of a fatty acid spin-probe. However, 10 mM-prilocaine induced a lipid phase separation at around 11 degrees C that was attributed to the lipids of the internal (cytosol-facing) half of the bilayer. It is suggested that prilocaine (10 mM) can selectively perturb the inner half of the bilayer of rat liver plasma membranes owing to its preferential interaction with the acidic phospholipids residing there.     

Alterations in the physicochemical properties of renal cortical membranes in rats with experimental cirrhosis of the liver.

Changes in the major component of renal cortical membranes as well as membrane fluidity and Na+, K+, ATPase activity have been studied in membranes from the renal cortex of rats with experimental liver cirrhosis, which show renal sodium and water retention, and in normal animals. Rats with cirrhosis of the liver show a decrease in cholesterol, phospholipid and protein content, without changes in cholesterol/phospholipid molar ratio. In addition there is a small decrease in 14:0 and 18:2 and an increase in 20:4 content, without differences in unsaturation degree. Membrane fluidity was decreased in renal membranes from cirrhotic rats when compared with normal ones. Na+, K+, ATPase activity was higher in cirrhotic than in normal renal membranes could be related with the changes in renal water and electrolyte changes shown by cirrhotic rats.     

Modulation of the in vitro activity of lysosomal phospholipase A1 by membrane lipids

Lysosomal phospholipases play a critical role for degradation of cellular membranes after their lysosomal segregation. We investigated the regulation of lysosomal phospholipase A1 by cholesterol, phosphatidylethanolamine, and negatively-charged lipids in correlation with changes of biophysical properties of the membranes induced by these lipids. Lysosomal phospholipase A1 activity was determined towards phosphatidylcholine included in liposomes of variable composition using a whole-soluble lysosomal fraction of rat liver as enzymatic source. Phospholipase A1 activity was then related to membrane fluidity, lipid phase organization and membrane potential as determined by fluorescence depolarization of DPH, 31P NMR and capillary electrophoresis. Phospholipase A1 activity was markedly enhanced when the amount of negatively-charged lipids included in the vesicles was increased from 10 to around 30% of total phospholipids and the intensity of this effect depended on the nature of the acidic lipids used (ganglioside GM1相似文献   

Studies on the purified, lipid-reconstituted (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes. Dependence of enzyme activity on lipid headgroup and hydrocarbon chain structure

The purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes was successfully reconstituted with a number of different phospho- and glycolipids, and the ability of these lipids to support the function of this enzyme was evaluated by their ability to increase the specific activity of the purified enzyme and by their ability to restore its lipid-phase state-dependent properties which were lost during purification. The incorporation of this ATPase into liposomes composed of the endogenous membrane lipids of the organism, or of zwitterionic phospholipids such as phosphatidylcholine or phosphatidylethanolamine, results in a full reconstitution of its activity and its lipid-phase state-dependent properties. In contrast, anionic phospholipids alone, or in combination with zwitterionic phospholipids at concentrations higher than 10 mol % of the anionic phospholipid, cause an irreversible inhibition of this ATPase. However, when combined with neutral glycolipids, larger amounts of anionic phospholipid can be tolerated without enzyme inhibition. Phosphatidylcholines with acyl chains of 14-24 linear carbon atoms and varying degrees of branching and unsaturation successfully reconstitute the enzyme, in marked contrast to the shorter chain homologues, which were ineffective. Our results indicate that the full expression of the activity of the A. laidlawii B ATPase requires a host lipid bilayer membrane of low to moderate negative surface charge which is predominantly liquid-crystalline and of a minimal bilayer thickness. Once such requirements are met, the enzyme exhibits considerable flexibility regarding the nature of the lipids which can effectively support its function. In particular, the activity of the A. laidlawii B ATPase is not very sensitive to lipid "fluidity" in the liquid-crystalline state.     

Changes in cardiac electrophysiology,morphology, tissue biochemistry and vascular reactions in glutathione depleted animals

Human placental syncytiotrophoblast basal membrane plays an important role in transfer of nutrients from the mother to the growing fetus all throughout gestation. The membrane lipid composition together with the bilayer fluidity is found to be the major index in modulation of these transport processes. In the present study, the effects of changing lipid composition on the placental basal membrane fluidity and the modulating influence of the latter on membrane enzyme and transport functions with progress of gestation,were investigated. Steady-state fluorescence analysis using 1,6-diphenyl-1,3,5 hexatriene as the probe, indicated a decrease in fluorescence anisotropy of both labeled native membrane vesicles and liposomes prepared from lipids extracted from the basal membrane vesicles, signifying increased bilayer fluidity with progress of gestation. This in turn, was successfully correlated to the lowering of cholesterol content and enhanced phospholipid concentration with a steady decrease in cholesterol/phospholipid ratio during placental development. Enhanced Na⁺-K⁺-ATPase activity and steady-state glucose uptake across basal membrane with gestational progress suggested modulation of membrane protein functions by the fluidity, which was further corroborated by the increased bilayer fluidity and enzyme activity in benzyl alcohol treated basal membrane in each gestational age group.     

Reconstitution of the leucine transport system of Lactococcus lactis into liposomes composed of membrane-spanning lipids from Sulfolobus acidocaldarius.

The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated. Liposomes were prepared from mixtures of monolayer lipids and the bilayer lipid phosphatidylcholine (PC), analyzed on their miscibility, and fused with membrane vesicles from L. lactis. Freeze-fracture electron microscopy demonstrates that the bipolar lipids in the hybrid membranes adopted a monomolecular organization at high S. acidocaldarius lipid content. Leucine transport activity (i.e., delta mu H(+)-driven and counterflow uptake) increased with the content of S. acidocaldarius lipids and was optimal at a one-to-one (w/w) ratio of PC to S. acidocaldarius lipids. Membrane fluidity decreased with increasing S. acidocaldarius lipid content. These data suggest that transport proteins can be functionally reconstituted into membranes composed of membrane-spanning lipids provided that membrane viscosity is restricted.     

Calcium alters the acyl chain composition and lipid fluidity of rat hepatocyte plasma membranes in vitro

Calcium ion decreases the lipid fluidity of isolated rat hepatocyte plasma membranes by modulating the activity of membrane enzymes which alter the lipid composition. To explore the mechanism of the effect of the cation, eight fluorophores were used to assess lipid fluidity via estimations of either steady-state fluorescence polarization or excimer fluorescence intensity. The results demonstrate that the reduction in fluidity occurs in the hydrophobic interior of the bilayer and that both the dynamic and static (lipid order) components of fluidity are affected by treatment with calcium. Analysis of the membrane lipids demonstrates that calcium treatment decreases the arachidonic acid content of the polar lipid fraction and, thereby, reduces the double-bond index of the fatty acids. This change in composition, which is expected to reduce the lipid fluidity, may result from activation by calcium of the endogenous hepatocyte plasma membrane phospholipase A2.     

Localization of cholesterol in the colonic epithelium of the guinea pig: regional differences and functional implications

Summary It is generally accepted that variations in membrane cholesterol content affect the fluidity of the bilayer, thus altering its permeability. In the biological membranes, in physiological conditions, a high cholesterol content rigidifies the bilayer decreasing its permeability, a lower cholesterol content induces the opposite effect by increasing the permeability. Since differences in the epithelial permeability for short chain fatty acids have previously been demonstrated in the proximal and distal colon of the guinea pig, these two regions were investigated to establish whether differences in membrane cholesterol content of the absorbing cells can be demonstrated. Freeze-fracture replicas of filipin-treated colonic tissue were used. The results show that in the proximal colon the density of filipin cholesterol complexes located on the luminal plasma membrane of the columnar absorbing cells was significantly higher (about twice) than in the distal colon. Therefore the lower amount of cholesterol present in the membrane of the absorbing cells in the distal colon indicates a greater fluidity of the membranes of the epithelial cells in this region. Such fluidity could be correlated to the higher absorption rates of shortchain fatty acids characteristic of this region.     

Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms

Steroid hormones are lipophilic suggesting they intercalate into the bilayer of target cell plasma membranes, potentially altering the fluidity and function of the membrane. The present study measured the effects of steroidal exposure on both phospholipid fluidity and integral protein mobility. Studies were performed on the effects of a variety of steroids on phosphatidylcholine liposomes, synaptosomal plasma membranes and sarcoplasmic reticulum membranes. Progesterone decreased the lipid fluidity, whereas testosterone had no effect on lipid movement. The estrogen, 17 beta-estradiol, an aromatised metabolite of testosterone, increased lipid mobility. In each case, the steroid action was concentration-dependent. The steroids all increased the activity of the Ca2+ ATPase of SR membrane, in keeping with their effects on this enzyme's aggregation state. The results suggest that, although lipid fluidity is a factor influencing protein activity, their mobility within the bilayer is the primary determinant of enzyme activity in the membrane for most proteins.     

Organization of lipids in sarcoplasmic reticulum membrane and Ca2+-dependent ATPase activity.

The organization of lipids in sarcoplasmic reticulum membrane was studied with a variety of stearic spin labels and a phosphatidylcholine spin label. The ESR spectra of the spin-labeled membranes consisted of two components, one due to labels in lipid bilayer structure and the other due to more immobilized labels. The relative intensity of the immobilized component increased when the lipid content of the membrane was decreased by treatment with phospholipase A [EC 3.1.1.4] and subsequent washing with bovine serum albumin. Membrane containing 30% of the intact phospholipid, i.e.0.15 mg of phospholipid per mg of protein, showed a spectrum consisting only of the immobilized component (the overall splitting ranged from 58.5 G to 60.5 G). The immobilized component was ascribed to lipids complexed with protein. The fraction of lipids in the two different organizations was determined from the ESR spectrum. The activity of the Ca2+-Mg2+ dependent ATPase [ATP phosphohydrolase, EC 3.6.1.3] was found to increase almost linearly with the lipid bilayer content in the membrane, whereas phosphoenzyme formation was almost independent of the bilayer content. This indicated that the bilayer structure is necessary for the ATPase to attain its full transport activity.     

Changes in membrane fluidity evoked by organophosphorus insecticide bromfenvinfos and its methylated analogue

The interaction of organophosphorus insecticides bromfenvinfos and methyl bromfenvinfos with model and native membranes was investigated by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe located in the hydrophobic core of the bilayer and 1,3-bis-(1-pyrene)propane, a probe distributed in the outer region of the bilayer. DPH reported a broadening of the transition profile and solidifying effects in the fluid phase of liposomes formed from dimyristoyl (DMPC), dipalmitoyl (DPPC), and distearoyl (DSPC) phosphatidylcholine in the presence of the insecticides. A shift of the transition temperature towards a lower temperature was observed in DPPC- and DSPC-bromfenvinfos-treated vesicles. Py(3)Py detected an ordering effect of the insecticides in the fluid state of the lipids and abolished pre-transition in DPPC and DSPC vesicles. These results suggest that the insecticides localize in the co-operative region of the bilayer. Cholesterol added to DMPC decreased the influence of the insecticides as reported by both DPH and Py(3)Py. The effect of the insecticides on the fluidity of some native membranes, namely erythrocytes, lymphocytes, brain microsomes, and sarcoplasmic reticulum, depended on the cholesterol content in these membranes, the higher the cholesterol content, the smaller the solidifying effect. The physical mechanism of action of the insecticides on membrane lipids can be similar to that of cholesterol. All observed effects were more pronounced for bromfenvinfos than for its methylated analogue which correlates with the toxicity of these compounds for mammals.     

Effect of lipid composition on rat liver nuclear membrane fluidity

Nuclear membrane fluidity is measured in rat liver by use of the fluorescence anisotropy of two probes: diphenylhexatriene and its cationic derivative trimethylammonium-diphenylhexatriene. It has been shown that, in 2-month-old rat liver cells, the bilayer surface is less fluid than the hydrophobic core. The fluidity was higher in 6-day-old rat liver nuclei, in which both the amount of cholesterol and the cholesterol/phospholipid ratio decreased. The influence of the single phospholipids, and in particular of phosphatidylcholine, has been studied by increasing the phosphatidylcholine with a choline base exchange reaction in isolated nuclear membranes. After this reaction, the fluorescence anisotropy of the bilayer surface increased, whereas at the hydrophobic core it decreased. Analysis of fatty acid composition shows an increase of phosphatidylcholine unsaturated fatty acids. The results show that the fluidity of nuclear membranes changes in relation to the lipid content and to the fatty acid composition. The role of nuclear membrane fluidity in cell function is discussed. © 1997 John Wiley & Sons, Ltd.     

Role of phosphatidylethanol in membranes. Effects on membrane fluidity, tolerance to ethanol, and activity of membrane-bound enzymes.

We investigated the effect of phosphatidylethanol (PEt) on fluidity and membrane tolerance to the fluidization induced by ethanol as well as on the activity of two membrane-bound enzymes, Na+/K+ ATPase and 5'-nucleotidase. PEt was synthesized from 1,2-dimyristoylphosphatidylcholine and phosphatidylcholine from bovine brain and studies were performed to determine the optimal experimental conditions for the insertion of PEt in natural bilayers. The effects of PEt, evaluated by differential scanning calorimetry or fluorescence polarization techniques, were studied in model membranes made of synthetic phospholipids or made of total lipids extracted from rat brain crude mitochondrial fraction (P2 fraction) and from natural membranes (P2 fraction). The presence of PEt increased the fluidity of artificial as well of natural membranes, but tolerance to the addition of ethanol, displayed by dimyristoylphosphatidylcholine vesicles and by natural membranes containing PEt, was lacking in vesicles made of dimyristoylphosphatidylethanolamine and in artificial bilayers reconstituted from total P2 lipid extracts, suggesting an involvement of PC on PEt-induced ethanol resistance. Na+/K+ ATPase activity was enhanced by the addition of small amounts of ethanol (up to 50 mM) and progressively inhibited at higher concentrations, while 5'-nucleotidase was not affected up to 400 mM ethanol. The presence of PEt in the bilayer exerted the opposite effects on the two enzymes, reducing the Na+/K+ ATPase activation induced by ethanol and enhancing 5'-nucleotidase activity. The mechanisms of the PEt-induced modifications are discussed.     

The Influence of Membrane Lipids in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Gamma-Hemolysins Pore Formation

The natural target of Staphylococcus aureus bicomponent γ-hemolysins are leucocyte cell membranes. Because a proteinaceous receptor has not been found yet, we checked for the importance of the different membrane lipid compositions by measuring the activity of the toxin on several pure lipid model membranes. We investigated the effect of membrane thickness, fluidity, and presence of nonbilayer lipids and found that the toxin pore-forming ability increased in the presence of phosphocholines with short saturated acyl chains or with unsaturated chains even though not short. An increase of activity was also evident in the presence of cone-shaped lipids like phosphatidylethanolamine or diphytanoylphosphatidylcholine, whereas cylindrical lipids, like sphingomyelin, did not favor the activity. All these results suggest that γ-hemolysins could bind to the bilayer only if the phosphatidylcholine (PC) head is freely accessible. This condition is satisfied by the concurrent presence of cholesterol and certain lipids, as highlighted by the so-called umbrella model (J. Huang and G. W. Feigenson, Biophys J 76:2142–2157, 1999). According to this model, cholesterol could help to a better exposition of PC head groups only if acyl chains are short or unsaturated. In fact, phosphatidylcholines with more than 13 carbon atoms acyl chains can cover cholesterol molecules; in this way, PC head groups pack tightly, rendering them inaccessible to the toxin, which thus shows a reduced pore-forming ability.     

Growth in excess copper induces changes in the lipid composition and fluidity of PSII-enriched membranes in wheat

Changes in the lipid composition and fluidity of PSII-enriched thylakoids were studied in seedlings of wheat ( Triticum durum Desf. cv. Adamello) grown in nutrient solution supplemented with CuSO₄ to achieve a final concentration of 10 and 50 μ M Cu. Metal content increased in the chloroplasts of the 50 μ M Cu-grown plants. PSII isolated from wheat supplied with 10 μ M Cu did not show any alteration in the lipid composition or in the lipid and protein levels of the membranes, nor was any change in the ultrastructure of the membranes detected. The 50 μ M Cu-grown plants showed thylakoid swelling, particularly in the stroma and terminal grana thylakoids. Furthermore, an alteration in the lipid composition of PSII preparations was observed together with a decrease in the lipid content, which resulted in a reduction in the lipid to protein ratio. The monogalactosyldiacylglycerol (MGDG) to digalactosyldiacylglycerol (DGDG) molar ratio decreased, whereas the degradation of the polar lipids caused an accumulation of free fatty acids (FFA). The total amount of unsaturated lipids associated with the PSII-enriched membranes of wheat was not affected by excess copper supplies, even though changes in the individual fatty acids occurred. The effect of copper on the fluidity of PSII membranes was evaluated by electron paramagnetic resonance (EPR) measurements, using spin-probed fatty acids as probes. The PSII membranes, spin probed by means of 5- and 16-doxylstearic acids, showed that only the fluidity of the surface region of the bilayer close to the polar head group was reduced following the 50 μ M Cu supply. In contrast, the fluidity of the inner membrane region of the bilayer did not show any change. The implications of changes in the lipid composition and lipid-protein interactions on the fluidity of specific transversal membrane regions are discussed.