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1.
Lipid-protein model membranes, prepared from bovine brain white matter and containing all the lipids and Folch-Lees proteolipids, have been studied in macroscopically oriented multibilayers. To examine the lipid environment the membranes were spin labeled with the cholestane spin label (3'-spiro(2'=(N-oxyl-4',4'-dimethyl-oxazolidine))5alpha-cholestane) and a fatty acid spin label (4',4'-dimethyloxazolidine-N-oxyl derivative of 5-ketostearic acid). The ESR spectra exhibit two components arising from fairly well oriented and completely unoriented lipids. Up to a temperature of 55 degrees C the amount of oriented lipids is almost constant, being about 35%. At higher temperatures this percentage drops rapidly to zero. It is shown that the presence of unoriented lipids arises mainly from disrupted areas in the lipid bilayer structure. This is confirmed by electron miccroscopy and from an analysis of the temperature dependence of the order parameters of the spin labels. The presence of locally disrupted lipid parts in the bilayer is discussed in relation to the interaction of the brain white matter lipids with Folch-Lees protein. 相似文献
2.
Studies of the influence of fatty acids, which were the component of intestinal mucosal lipids, on the permeability of several drugs across bilayer lipid membranes generated from egg phosphatidylcholine and intestinal lipid have been pursued. The permeability coefficients of acid, salicylic acid and acid (anionic-charged drug) increased when fatty acids such as lauric, stearic, oleic, linoleic and linolenic acid were incorporated into the bilayer lipid membranes generated from phosphatidylcholine. In the presence of methyl linoleate and oleyl alcohol, no enhancing effect on acid transfer was obtained. The effect of fatty acids was more marked at pH 6.5 than at pH 4.5. In contrast, upon the addition of fatty acids to intestinal lipid membranes which originally contained fatty acids, the permeability coefficient of acid tended to decrease, though the permeability through intestinal lipid membranes was larger than that of phosphatidylcholine membranes. The permeability of acid across bilayer lipid membranes from intestinal phospholipids was significantly decreased to about equal that of phosphatidylcholine membranes, and reverted to the value of intestinal lipid membranes when fatty acids were added to intestinal phospholipids. It seemed reasonable to assume that free fatty acids in the intestinal neutral lipid fraction could contribute to the increase in the permeability of acid. On the basis of above results, possible mechanisms for good absorbability of weakly acidic drugs from the intestine are discussed. 相似文献
3.
Davor Juretić 《生物化学与生物物理学报:生物膜》1977,469(2):137-150
By using the choline starvation process it is possible to deplete the membranes of Neurospora crassa choline auxotroph of phosphatidylcholine, without affecting the viability of germinated spores or whole mycelium. Spin label probes were used to examine the possible dependence of the physical state of cellular lipids on the presence of phosphatidylcholine in the membranes.Increased freedom of rotational motion of lipid soluble probes was regularly detected in choline-starved mycelium. The accumulation of neutral lipids (mostly triglycerides) in bulk form was also observed during the choline starvation process. The experiments with isolated and separated lipid classes indicated that the observed increase in fluidity of lipids in choline-starved mycelium is partly due to the difference in physical properties between bulk lipids and membrane lipids. Spin label probe 2N4 (), which can partition at the membrane-water interface, exhibited easier partitioning among membrane lipids of choline-starved mycelium. 相似文献
4.
Neutron diffraction studies on phosphatidylcholine model membranes. I. Head group conformation 总被引:19,自引:0,他引:19
Neutron diffraction experiments on selectively deuterated lipids provide a new method of determining to a segmental resolution the mean conformation of a lipid molecule as projected along the bilayer normal, despite the high amount of disorder that exists in these bilayers. In addition, a time-averaged picture of the extent of the positional fluctuations of the individual segments in this direction can be given. This is demonstrated for a multilamellar system of bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In this paper the head group region of the molecule is examined and this carries the zwitterionic phosphocholine group that determines the electrostatic interaction in the bilayer. Samples deuterated at four different positions in the head group region were measured as oriented samples at 6% () water content at 20 °C (Lβ′ phase) and at 10% () at 70 °C (Lα phase) and as unsonicated dispersions with 25% () water at 28 °C (Lβ′ phase) and 50 °C (Lα phase). From the oriented samples, reflections up to ten orders, and from the powder type samples only four orders, were collected. The derived structure factors for the deuterated segments were fitted assuming a Gaussian distribution of the segments along the bilayer normal. The mean label position was determined for each label under different conditions of water content and temperature with a precision of better than ± 1 ångström in most cases. The data clearly show that the average orientation of the zwitterionic phosphocholine group is almost parallel to the membrane surface in the gel state (Lβ′) as well as in the liquid crystalline state (Lα). It is interesting to note that in a recent dielectric investigation on this multilamellar system at 25% () water content the same mean orientation of the dipole was found (Shepherd &; Büldt, 1978). 相似文献
5.
The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with . This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the enzyme and may reflect a protein-directed phospholipid specificity for this enzyme. 相似文献
6.
Michael J. Barber Andrew S. Zektzer Gerald M. Rosen Helen A. Demos Elmer J. Rauckman 《生物化学与生物物理学报:生物膜》1984,776(1):159-168
Hepatic microsomal membranes, prepared under various conditions that yield either ‘intact’ or ‘disrupted’ microsomal vesicles, have been labeled via the sulfhydryl groups of intrinsic membrane proteins using nitroxide analogs of . Electron paramagnetic resonance spectra revealed the presence of two dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which were quantitated using a parameter designated the ‘’ ratio. For latent microsomes, the value of this parameter was determined to be and was influenced by factors such as label/protein ratio, incubation period, nitroxide structure, temperature and pH. The ratio was also sensitive to the degree of membrane integrity as revealed by the latency of mannose 6-phosphate activity of glucose-6-phosphohydrolase. In addition, membrane disruption resulted in a corresponding decrease in the order parameter for nitroxide-labeled fatty acids intercalated within the lipid bilayer. The ratio was observed to be dependent upon the method of microsome preparation yielding values of for ‘hypertonically disrupted’ vesicles and for ‘mechanically disrupted’ vesicles. Microsomal marker enzymes such as cytochrome and FAD-containing monooxygenase retained significant levels of functionally following nitroxide incorporation. 相似文献
7.
Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe3+, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 °C, while that for phosphatidylserine spin label had only one transition at 30 °C.When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogeneous fluidity. Mg2+ or prevented the hemolysis-induced spectral changes. Ca2+ did not prevent the homogenization and acted antagonistically to Mg2+. The heterogeneity preservation by Mg2+ was nullified by trypsin, pronase or added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg2+ was dependent on hemolysis temperature, starting to decrease at 18 °C and vanishing at 40 °C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity. 相似文献
8.
M Okamoto R Miura Y Miyake T Yamano M Fujioka 《Biochemical and biophysical research communications》1979,87(1):300-307
Lipid bilayer of rat liver microsomes was spin-labeled by incubating with liposomes of 1-acyl 2-(12-doxylstearoyl) glycero-3-phosphorylcholine. When NADPH was added to the labeled microsomes, there appeared a rapidly tumbling component of spin label in the EPR spectrum. NADH was less effective than NADPH. The appearance of the sharp signal was prevented under anaerobic conditions or in the presence of either carbon monoxide, phenyl isocyanide or cytochrome . The appearance of the rapidly tumbling component in the EPR spectrum was found to be due to the release of spin moiety from the membrane into the aqueous phase. That the release was associated with superoxide anion formation or with lipid peroxidation is unlikely, since 1) superoxide dismutase had little effect, 2) addition of either α-tocopherol or EDTA did not inhibit the release. These observations suggest that electron transfer from NADPH to oxygen cytochrome P-450 system induces a physical perturbation in the lipid bilayer resulting in the release of its component into the aqueous phase. 相似文献
9.
The properties of mixtures of phosphatidylcholine and analogues of cholesterol bearing side chains of varying lengths were examined by a variety of methods. The incorporation of the analogues into sonicated liposomes and their effect on the rate of osmotic shrinking of multilamellar liposomes were determined. The ordering of a steroid spin label was studied in an oriented multibilayer system and the effect of the analogues on the phase transition of dipalmitoyl phosphatidylcholine monitored using the spin label TEMPO (). Mixtures of analogues and phospholipid were also studied in monolayers.In all the bilayer systems studied cholesterol caused the greatest ‘rigidifying’ effect, the analogues with shorter or longer side chains being less effective. However, in the monolayer experiments the length of the sterol molecule was found to be much less critical. It is suggested that cholesterol is anchored in position in a phospholipid bilayer by virtue of the molecule being the precise length required to maximise interactions between neighbouring molecules without disturbing the bilayer structure. 相似文献
10.
Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases 总被引:54,自引:0,他引:54
This work describes the structure of a variety of lecithin-water phases observed below the “melting” temperature of the hydrocarbon chains, with special emphasis on the conformation of the chains. The lecithins studied in this work are the homologous series dioctanoyl to distearoyl, 2-decanoyl-1-stearoyl, and a preparation from hen eggs. The hydrocarbon chains are found to adopt a variety of conformations in addition to type α, the liquid-like organization observed above the melting temperature. Type β: the chains are stiff and parallel, oriented at right angles to the plane of the lamellae and packed with rotational disorder in a two-dimensional hexagonal lattice (. Type β′: similar to β, but with the chains tilted with respect to the normal to the lamellae. Type δ: the chains are probably coiled into helices, whose axes are perpendicular to the plane of the polar groups and are packed with rotational disorder in a two-dimensional square lattice (, α is the predominant conformation, common to most lipids in the presence of water and at sufficiently high temperature, and the one more relevant to membranes; β is observed at lower temperatures in lipids whose chains are heterogeneous and in the presence of very small amounts of water; β′ is found in synthetic lecithins with identical chains, in the presence of variable amounts of water; δ is observed in dry lecithins. A highly ordered crystalline phase, yet displaying rotational disorder of the chains, is observed in almost dry lecithins. Most of the phases are lamellar, and contain one lipid bilayer per repeat unit. Two phases display two-dimensional lattices: Pδ, formed by ribbon-like elements with the chains in the δ conformation; Pβ′, formed by lamellae of type β′ distorted by periodic ripples. The results emphasize the clear-cut difference between the liquid-like and the other types of partly ordered conformations, as well as the correlations which exist between the chemical composition and the structure of the lipids below the melting temperature of the chains. 相似文献
11.
Glycoproteins in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups by galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components were fractionated on diatrizoate to give four synaptosomal membrane fractions (A to D) and a mitochondrial pellet (E). Fractions A and B represent highly purified synaptosomal plasma membranes. After separation of their polypeptides by electrophoresis, of the label was present in two bands: one about 72 000 and the other between 7800 and 3200 daltons. Seven other bands were labeled to various degrees: 160 000, 96 000, 53 000, 39 000, 34 000, 23 000 and 16 000 daltons. With isolated membranes (which incorporate 5–6 times more label) of label was present in polypeptides in three ranges: 160 000–96 000, 70 000–40 000 and 7800-3200. The number of polypeptides that can be labeled by treatment of isolated membranes is very large. In comparison, glycoproteins whose topographical distribution permits interaction with large molecules at the synaptic surface are very limited. It is further suggested that the external synaptosome membrane involves a relatively tight network of interacting molecules that cannot be readily penetrated by large molecules. 相似文献
12.
Mixtures of lipids and proteins, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, , of the lipid and aggregated below . For mixtures af dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains was shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid lipids were present.In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50–200 nm in length, around smooth patches of lipid.Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperarture of the lipid is discussed. 相似文献
13.
Charge-pulse current-relaxation studies have been performed with lipid bilayer membranes in the presence of the hydrophobic ion dipicrylamine. From the analysis of the relaxation times and amplitudes the translocation rate constant of dipicrylamine as well as the partition coefficient β between membrane surface and water could be evaluated. In a first series of experiments membranes made from monoolein or dioleoylphosphatidylcholine in a number of different solvents were studied, as well as virtually solvent-free bilayer membranes made from monolayers. The thickness of the hydrocarbon layer of these membranes varied between 5.0 and 2.5 nm. While β was almost insensitive to variations in , a strong decrease of with increasing membrane thickness was found; the observed dependence of on approximately agreed with the theoretically expected influence of membrane thickness on the height of the dielectric barrier. No specific differences between Mueller-Rudin films and solvent-free (Montal-Mueller) membranes other than differences in thickness were found. In a further series of experiments the chemical structure of the lipid was systematically varied (number and position of double bonds in the hydrocarbon chain, nature of the polar head group). The translocation rate constant was much larger in phosphatidylethanolamine membranes than in phosphatidylcholine membranes. A strong increase of was found when the number of double bonds in the hydrocarbon chain was increased from one to three. These changes were discussed in terms of membrane fluidity and dielectric barrier height. Much higher values of were observed in lipids with ester linkage between hydrocarbon chain and glycerol backbone, as compared with the corresponding ether analogs. This finding is qualitatively consistent with determinations of dipolar potentials in monolayers of ester and ether lipids. When cholesterol is added to phosphatidylcholine membranes, the translocation rate constant increases up to five-fold, while the partition coefficient β remains virtually constant. The variation of in this case can be largely accounted for by a decrease in membrane thickness and a concomitant reduction in dielectric barrier height. In membranes made from the negatively charged lipid phosphatidylserine the partition coefficient of dipicrylamine strongly increased with ionic strength, as expected from the Gouy-Chapman theory of the surface potential. 相似文献
14.
The effects of lateral tension on the properties of membranes are often explained in comparison with analogous experiments on monolayers, which yield more detailed data. To calculate the effects of changes in tension on the composition of, or incorporation of amphiphiles into membranes we examine (i) the fidelity of the monolayer analogy, (ii) the range of possible tensions in a membrane, and the way in which tensions arise and (iii) the equilibrium partitioning of amphiphiles between aqueous solution and a bilayer under tension. We argue that, at the same areas per molecule, a monolayer at an interface is a closer analogy of the lipid bilayer than a monolayer at an air/water interface. Next, we show from a thermodynamic argument that changes in membrane tension can affect the absorption of very large amphiphiles such as proteins, but that physiological tensions are unlikely to affect the absorption of lipids or drugs. Finally we consider the possibility that the measured bulk tension in a complicated membrane such as that of the erythrocyte may be larger than the local tension in the fluid mosaic portions, and suggest a model which explains the ability of the erythrocyte membrane to withstand much higher tensions than other biological membranes and lipid bilayers. 相似文献
15.
C. Mombers P.W.M. Van Dijck L.L.M. Van Deenen J. De Gier A.J. Verkleij 《生物化学与生物物理学报:生物膜》1977,470(2):152-160
Using differential scanning calorimetry and freeze fracture electron microscopy interactions were studied between lipids and a spectrin · action complex isolated from human erythrocyte membranes. With dispersions of , and mixtures of these two compounds, which for experimental reasons were chosen as the lipid counterpart, such an interaction could clearly be deduced from changes in the temperature and the enthalpy of the phase transition. Furthermore it was demonstrated that the interaction with this membrane protein protects the bilayer against the action of Ca2+ and Mg2+ and prevents fusion of lipid vesicles which easily occurs in some of the systems when divalent ions were added to the pure lipid vesicles. 相似文献
16.
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18.
Membrane lipid fluidity as rate limiting in the concanavalin A-mediated agglutination of pyBHK cells
The initial rate of concanavalin A-mediated agglutination of polyoma transformed Baby Hamster Kidney (pyBHK) cells follows Arrhenius kinetics. There is a smooth decrease in the agglutination rate from 37°C to 22°C with an activation energy of in this region. There is a sharp decrease in agglutination rate below 22°C. The addition of 0.1 mM 1,3-di-tert-2-hydroxyl-5-methylbenzene, a lipid perturber, increases the agglutination rate by a factor of two and increases the membrane lipid fluidity as determined by the spin label method. The rotational correlation time of the spin label 2N14 ) was measured. The sum of the enthalpy of activation of rotational diffusion and the enthalpy of activation of translational diffusion is very nearly equal to the enthalpy of activation of agglutination. This is consistent with the rate limiting step of agglutination being receptor diffusion, which is probably limited in pyBHK cells by membrane lipid fluidity. 相似文献
19.
Direct measurement of the partition coefficient of into phosphatidylcholine and phosphatidylcholine-cholesterol bilayers showed that (a) isotropic liquids are not good models for lipid bilayers and (b), Regular Solution Theory cannot, in general, be applied to lipid bilayer membranes at temperatures above their phase transition. Theoretical and experimental evidence is given. 相似文献
20.
Basolateral membranes isolated from hog kidney cortex, enriched 12- to 15-fold in (Na+ + K+)-ATPase activity, were 80% oriented inside-out as determined by assay of oubain-sensitive (Na+ + K+)-ATPase activity before and after opening of the membrane vesicle preparation with a mixture of deoxycholate and EDTA. In these membrane preparations 80% of total phosphatidylethanolamine was accessible to trinitrophenylation by trinitrobenzenesulfonic acid at 4°C, while at 37°C all of phosphatidylethanolamine fraction was chemically modified. Phospholipase C treatment resulted in hydrolysis of 80% phosphatidylethanolamine, 40% phosphatidylcholine and 35% of phosphatidylserine. Sphingomyelinase treatment resulted in 20% hydrolysis of sphingomyelin, presumably derived from right-side-out oriented vesicles. Results indicate that phosphatidylethanolamine is oriented exclusively on the outer leaflet of the lipid bilayer of inside-out oriented vesicles. Methylation of phospholipids in basolateral membranes with resulted in the three successive methylation of ethanolamine moiety of phosphatidylethanolamine to phosphatidylcholine. The for was 1·10?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg2+ was without any effect between pH 5 and 10. Basolateral membranes incubated in the presence of methyl donor, , exhibited increased (12–15%) (Ca2+ + Mg2+)-ATPase activity and increased ATP-dependent uptake of calcium. ATP-dependent calcium uptake in these vesicles was insensitive to oligomycin and ouabain but was abolished completely by 50 μM vanadate. The increase in ATP-dependent calcium uptake was due to an increase in and not due to a change in for Ca2+. Preincubation of membranes with , a methyltransferase inhibitor, abolished the stimulatory effect of phospholipid methylation on calcium uptake. Phospholipid methylation at both low and high pH did not result in a change in bulk membrane fluidity as determined by the fluorescence polarization of diphenylhexatriene. These results suggest that phospholipid methylation may regulate transepithelial calcium flux in vivo. 相似文献