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1.
Several barrier epithelia such as renal collecting duct, urinary bladder, and gastric mucosa maintain high osmotic pH and solute gradients between body compartments and the blood by means of apical membranes of exceptionally low permeabilities. Although the mechanisms underlying these low permeabilities have been only poorly defined, low fluidity of the apical membrane has been postulated. The solubility diffusion model predicts that lower membrane fluidity will reduce permeability by reducing the ability of permeant molecules to diffuse through the lipid bilayer. However, little data compare membrane fluidity with permeability properties, and it is unclear whether fluidity determines permeability to all, or only some substances. We therefore studied the permeabilities of a series of artificial large unilamellar vesicles (LUV) of eight different compositions, exhibiting a range of fluidities encountered in biological membranes. Cholesterol and sphingomyelin content and acyl chain saturation were varied to create a range of fluidities. LUV anisotropy was measured as steady state fluorescence polarization of the lipophilic probe DPH. LUV permeabilities were determined by monitoring concentration-dependent or pH-sensitive quenching of entrapped carboxyfluorescein on a stopped- flow fluorimeter. The relation between DPH anisotropy and permeability to water, urea, acetamide, and NH3 was well fit in each instance by single exponential functions (r > 0.96), with lower fluidity corresponding to lower permeability. By contrast, proton permeability correlated only weakly with fluidity. We conclude that membrane fluidity determines permeability to most nonionic substances and that transmembrane proton flux occurs in a manner distinct from flux of other substances.  相似文献   

2.
Influence of membrane physical state on the proton permeability of isolated lysosomes was assessed by measuring the membrane potential with 3,3′-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Changes in the membrane order were examined by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Both the membrane potential and proton leakage increased with fluidizing the lysosomal membranes by benzyl alcohol and decreased with rigidifying the membranes by cholesteryl hemisuccinate. The proton permeability increased to the maximum of 42% by the benzyl alcohol treatment and decreased to the minimum of 38.1% by the cholesteryl hemisuccinate treatment. Treating the lysosomes with protonophore CCCP increased the proton permeability by 58%. The effects of the membrane fluidization and rigidification can be reversed by rigidifying the fluidized membranes and fluidizing the rigidified membranes, respectively. The results indicate that the proton permeability of lysosomes increased and decreased with increasing and decreasing their membrane fluidity, respectively. Moreover, the lysosomal proton permeability did not alter further if the changes, either an increase or a decrease, in the fluidity exceeded some amount. The results suggest that the proton permeability of lysosomes can be modulated finitely by the alterations in their membrane physical state. Received: 27 September 1999 / Revised: 27 December 1999  相似文献   

3.
The effect of cardiolipin on the proton permeability of dipalmitoyl-phosphatidylcholine small unilamellar vesicles was examined by utilizing the pH-dependent fluorescence emission of 5- (and 6-) carboxyfluorescein. It has been found that the proton permeability of the phospholipid bilayer was greatly enhanced in the presence of cardiolipin, an acidic phospholipid mainly found in the inner mitochondrial membranes. In the presence of bovine heart cardiolipin, the bilayer surface hydration, as assessed with the fluorescence lifetime of 1-anilinonaphthalene-8-sulfonic acid, was increased, while hydration in the acyl chain region was not altered. In addition, the bilayer fluidity was also not affected. Taken together, these results suggest that the lipid-water interface is the major energy barrier for proton permeation of the bilayer vesicles, and alteration to properties of this interface by cardiolipin headgroup appears to be responsible for the enhanced proton permeability.  相似文献   

4.
The physical state of the lysosomal membrane was modulated with the membrane fluidizers n-propanol and n-octanol and with the membrane rigidifiers cholesteryl hemisuccinate and cholesterol. Membrane fluidity was examined by the steady-state fluorescence anisotropy of 2-(9-anthroyloxy) palmitic acid and 16-(9-anthroyloxy) palmitic acid. Fluidizing the membranes at the surface and center areas increased the proton permeability coefficient by 92.8 and 18.0%, respectively. Rigidifying the membranes at the surface and center areas decreased the coefficient by 68.2 and 40.2%, respectively. Proton leakage of the lysosomes increased and decreased similar to the coefficient changes with the treatments. The results indicate that lysosomal proton permeability is affected by its membrane's physical state, and the physical state of the membrane surface area affects the proton permeability more markedly. The proton permeability coefficient of liposomes was similar to that of lysosomes, suggesting that efflux of lysosomal protons might occur through the lipid part of the bilayer but not transmembrane proteins.  相似文献   

5.
Lysosomal permeability to potassium ions is an important property of the organelle. Influence of the membrane physical state on the potassium ion permeability of isolated lysosomes was assessed by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and monitoring the lysosomal proton leakage with p-nitrophenol. The membrane fluidity of lysosomes was modulated by treatment with membrane fluidizer benzyl alcohol and rigidifier cholesteryl hemisuccinate. Changes in the membrane order were examined by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The measurements of membrane potential and proton leakage demonstrated that the permeability of lysosomes to potassium ions increased with rigidification of their membranes by cholesteryl hemisuccinate treatment at 37 degrees C, and decreased with fluidization of their membranes by benzyl alcohol treatment at 2 degrees C. The changes in ion permeability could be recovered by fluidizing the rigidified membranes and rigidifying the fluidized membranes. The results suggest that the physical states of lysosomal membranes play an important role in the regulation of their K(+) permeability.  相似文献   

6.
Summary Proton and calcium permeability coefficients of large unilamellar vesicles made from natural complex mixtures of phospholipids were measured in various conditions and related to membrane fluidity. Permeability coefficients at neutral pH and 25°C were in the range of 104 cm sec1 and 2.5×1011 cm sec1 for protons and calcium, respectively. With the exception of two cases. (H+)>104 m and (Ca2+)>103 m, fluidity increases correspond to permeability increases. Theoretical analysis shows that, for both ions, the measured values of permeability coefficients imply that the permeation process is controlled by the productD 1 D 2 of the diffusion coefficient from the medium into the membrane (D 1) by the diffusion coefficient in the membrane (D 2). Further analysis ofD 1 values deduced from combined use of permeability and fluidity data shows that the solubilization should occur in a medium of dielectric constant of about 12, suggesting the involvement of the hydration water of membranes. High proton concentrations, although having virtually no effect on fluidity, trigger the appearance of (i) lateral heterogeneity in membranes, as seen by31P NMR, and (ii) large permeability increases. It is proposed that the main effect of fluidity and/or lateral heterogeneity on permeability may bevia the membrane hydration control. We conclude that the current assumption that permeability is controlled by fluidity should be regarded with caution, at least in the case of ions and natural mixtures of phospholipids.  相似文献   

7.
The biochemical and biophysical properties of the membrane and some general characteristics of the response of Lactobacillus casei ATCC 393 (reclassified Lactobacillus zeae) to hyperosmotic conditions were studied. Under hypertonic conditions, the hydrophobicity and the bile salt sensitivity of the cultures were increased. The glycolipid AcylH3DG is only present in membranes of NaCl containing medium, whereas, H4DG undergoes a significant increment and H2DG a significant decrease. The fluidity of both the purified membranes and the total lipid vesicles, as determined with the fluorescent probe DPH, did not change in conditions of high salinity. This was coincident with changes in the fatty acid (FA) composition where an increase in the saturated/unsaturated FA ratio was compensated by a rise in the fluidifying 11,12-methyleneoctadecanoic FA (cyc 19:0). Under osmotic stress conditions, Laurdan and acridine orange in total lipid vesicles showed increased lateral lipid packing and proton permeability, respectively.  相似文献   

8.
Bilayer asymmetry in the apical membrane may be important to the barrier function exhibited by epithelia in the stomach, kidney, and bladder. Previously, we showed that reduced fluidity of a single bilayer leaflet reduced water permeability of the bilayer, and in this study we examine the effect of bilayer asymmetry on permeation of nonelectrolytes, gases, and protons. Bilayer asymmetry was induced in dipalmitoylphosphatidylcholine liposomes by rigidifying the outer leaflet with the rare earth metal, praseodymium (Pr3+). Rigidification was demonstrated by fluorescence anisotropy over a range of temperatures from 24 to 50 degrees C. Pr3+-treatment reduced membrane fluidity at temperatures above 40 degrees C (the phase-transition temperature). Increased fluidity exhibited by dipalmitoylphosphatidylcholine liposomes at 40 degrees C occurred at temperatures 1-3 degrees C higher in Pr3+-treated liposomes, and for both control and Pr3+-treated liposomes permeability coefficients were approximately two orders of magnitude higher at 48 degrees than at 24 degrees C. Reduced fluidity of one leaflet correlated with significantly reduced permeabilities to urea, glycerol, formamide, acetamide, and NH3. Proton permeability of dipalmitoylphosphatidylcholine liposomes was only fourfold higher at 48 degrees than at 24 degrees C, indicating a weak dependence on membrane fluidity, and this increase was abolished by Pr3+. CO2 permeability was unaffected by temperature. We conclude: (a) that decreasing membrane fluidity in a single leaflet is sufficient to reduce overall membrane permeability to solutes and NH3, suggesting that leaflets in a bilayer offer independent resistances to permeation, (b) bilayer asymmetry is a mechanism by which barrier epithelia can reduce permeability, and (c) CO(2) permeation through membranes occurs by a mechanism that is not dependent on fluidity.  相似文献   

9.
The ATP synthase of chloroplasts consists of the proton channel, CF0, and the catalytic part, CF1, which carries nucleotide-binding sites on subunits alpha and beta. The still poorly understood interaction between CF0 and the catalytic sites on CF1 is mediated by the smaller subunits gamma, delta and epsilon of CF1. We investigated the ability of purified delta to block proton leakage through CF0 channels after their exposure by removal of the CF1 counterpart. Thylakoids were partially depleted of CF1 by EDTA treatment. This increased their proton permeability and thereby reduced the rate of photophosphorylation. Subunit delta was isolated and purified by FPLC [Engelbrecht, S. and Junge, W. (1987) FEBS Lett. 219, 321-325]. Addition of delta to EDTA-treated thylakoids reconstituted high rates of phenazine-methosulfate-mediated photophosphorylation. Since delta does not interact with nucleotides by itself, the reconstitution was due to a reduction of the proton leakage through open CF0 channels. The molar ratio of purified delta over exposed CF0, which started to elicit this effect, was 3:1. However, if delta was added together with purified CF1 lacking delta, in a 1:1 molar ratio, the relative amount over exposed CF0 was as low as 0.06. This corroborated our previous conclusion [Lill, H., Engelbrecht, S., Sch?nknecht, G. and Junge, W. (1986) Eur. J. Biochem. 160, 627-634] that only a very small fraction of exposed CF0 was actually proton-conducting but with a very high unit conductance. CF1 including delta was apparently rebound preferentially to open CF0 channels. Although the ability of delta to control proton conduction through CF0 was evident, it remains to be established whether delta acts as a gated proton valve or as a conformational transducer in the integral CF0CF1 ATPase.  相似文献   

10.
We have confirmed that the respiration rate of rat liver mitochondria can be substantially inhibited with only a small drop in proton motive force. We have directly measured the passive proton permeability as a function of delta psi by using K+ diffusion potentials and have shown that there is a large increase in proton permeability at high delta psi. This can quantitatively account for the inhibitor titrations of respiration. delta psi and delta pH were shown to have roughly equal effects on the relatively high respiration rate in static head. The permeabilities to K+, tetramethylammonium+ and choline+ were shown to increase greatly at high delta psi, in a similar way to proton permeability, indicating a similar mechanism of entry.  相似文献   

11.
H+/OH- permeation through lipid bilayers occurs at anomalously high rates and the determinants of proton flux through membranes are poorly understood. Since all life depends on proton gradients, it is important to develop a greater understanding of proton leak phenomena. We have used stopped-flow fluorimetry to probe the influence of two lipid raft components, chol (cholesterol) and SM (sphingomyelin), on H+/OH- and water permeability. Increasing the concentrations of both lipids in POPC (palmitoyl-2-oleoyl phosphatidylcholine) liposomes decreased water permeability in a concentration-dependent manner, an effect that correlated with increased lipid order. Surprisingly, proton flux was increased by increasing the concentration of chol and SM. The chol effect was complex with molar concentrations of 17.9, 33 and 45.7% giving 2.8-fold (P<0.01), 2.2-fold (P<0.001) and 5.1-fold (P<0.001) increases in H+/OH- permeability from a baseline of 2.4x10(-2) cm/s. SM at 10 mole% effected a 2.8-fold increase (P<0.01), whereas 20 and 30 mole% enhanced permeability by 3.6-fold (P<0.05) and 4.1-fold respectively (P<0.05). Supplementing membranes containing chol with SM did not enhance H+/OH- permeability. Of interest was the finding that chol addition to soya-bean lipids decreased H+/OH- permeability, consistent with an earlier report [Ira and Krishnamoorthy (2001) J. Phys. Chem. B 105, 1484-1488]. We speculate that the presence of proton carriers in crude lipid extracts might contribute to this result. We conclude that (i) chol and SM specifically and independently increase rates of proton permeation in POPC bilayers, (ii) domains enriched in these lipids or domain interfaces may represent regions with high H+/OH- conductivity, (iii) H+/OH- fluxes are not governed by lipid order and (iv) chol can inhibit or promote H+/OH- permeability depending on the total lipid environment. Theories of proton permeation are discussed in the light of these results.  相似文献   

12.
The effects of two insecticides isomers, α- and β-endosulfan, on the passive proton permeability of large unilamellar vesicles (LUV) reconstituted with dipalmitoylphosphatidylcholine (DPPC) or mitochondrial lipids were reported. In DPPC (LUV) gel phase, at 30 °C, the global kinetic constant (K) of proton permeability (proportional to the proton permeability) initially increased slightly with the increase of α-endosulfan/lipid molar ratio up to 0.143. In the range from 0.143 to 0.286, a discontinuity in the increment occurred and, above this range, the proton permeability increased substantially. In DPPC fluid phase, at 48 °C, the proton permeability showed a behavior identical to that observed in gel DPPC, with a sharp increase for α-endosulfan/lipid molar ratios ranging from 0.143 to 0.286. At these and higher concentrations, α-endosulfan induced phase separation in the plane of DPPC membranes, as revealed by differential scanning calorimetry (DSC). Conversely to α-endosulfan, β-endosulfan induced only a slight increase in the proton permeability, either in the fluid or the gel phase of DPPC, for all β-endosulfan/lipid molar ratios tested. Additionally, the effects of the endosulfan isomers on the proton permeability of mitochondrial fluid lipid dispersions, at 37 °C, are similar to those described for DPPC. The β-isomer induced a very small effect, and α-endosulfan, at low concentrations, increased slightly the proton permeability, but for insecticide/lipid molar ratios above 0.143 the permeability increased substantially. Consequently, the membrane physical state of synthetic and native lipid dispersions, as affected by the structural features of α- and β-endosulfan, influenced the proton permeability. The effects here observed in vitro suggest that the formation of lateral membrane domains may underlay the biological activity of α-endosulfan in vivo, contributing to its higher degree of toxicity as compared with β-endosulfan.  相似文献   

13.
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.  相似文献   

14.
Abstract: In contrast with most other lipid substrates, in this article we show that liposomes prepared from the total myelin lipids exhibited a negligible proton permeability. Neither the generation of valinomycin-induced potassium diffusion potentials as high as -177 mV nor the imposition of large pH gradients (up to three units) was able to produce a substantial flux of protons through liposomal membranes, as determined by the distribution of [14C]-methylamine, or the changes in the fluorescence of the probes 9-aminoacridine, acridine orange, and pyranine. The presence of cations (Na+, K+, Ca2+) did not alter this behavior. Voltage clamping did not increase the trans-membrane ApH-driven proton permeability. However, II-posome diameter was found to be critical because small unilamellar vesicles displayed a much higher proton permeability than large unilamellar or multilamellar vesicles. This abnormally low proton permeability is interpreted by virtue of the characteristic biochemical composition of myelin lipid matrix, with a high content of cholesterol and sphingolipids and a very low level of free fatty acids. These results could be important for elucidating the role of myelin in the regulation of pH in the brain. In addition, the myelin lipid extract could be useful for reconstituting proteins that participate in the transport of H+ through the membrane.  相似文献   

15.
Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions. In some thermophiles, sodium is the sole energy-coupling ion. To test whether sodium is the preferred coupling ion at high temperatures, the proton- and sodium permeability was determined in liposomes prepared from lipids isolated from various bacterial and archaeal species that differ in their optimal growth temperature. The proton permeability increased with the temperature and was comparable for most species at their respective growth temperatures. Liposomes of thermophilic bacteria are an exception in the sense that the proton permeability is already high at the growth temperature. In all liposomes, the sodium permeability was lower than the proton permeability and increased with the temperature. The results suggest that the proton permeability of the cytoplasmic membrane is an important parameter in determining the maximum growth temperature.  相似文献   

16.
On the nature of the mitochondrial proton leak.   总被引:2,自引:0,他引:2  
Respiring mitochondria have a significant passive permeability to protons; the mechanism of this proton leak is unknown. Several putative mechanisms were tested. Mitochondrial permeability to small sugars was unaffected by energization, suggesting that there is no significant dielectric breakdown at high membrane potential. Mitochondria are argued to have a proton permeability that is 6 to 8 orders of magnitude higher than the permeability to other cations, suggesting that the proton leak is probably not via a simple pore or membrane defect. 15-30% of the proton leak of freshly prepared mitochondria was extractable with bovine serum albumin and is probably due to fatty acids. Little if any of the proton leak appears to be due to cycling of ions other than protons, or to be associated with the functional activity of the proton pumps. The mitochondrial proton leak shares several properties with the proton permeability of pure phospholipid bilayers, suggesting that they share the same mechanism, although the leak through the bilayer in mitochondria may be modified by the presence of proteins.  相似文献   

17.
The ATP-generated proton pumping across tonoplast vesicles from chilling-sensitive Boro rice (Oryza sativa L. var. Boro) cultured cells was markedly decreased by chilling at 5 degrees C for 3 d. The membrane fluidity of core hydrophobic and surface hydrophilic regions of the lipid bilayer was measured by steady-state fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium 1,6-diphenyl-1,3,5-hexatriene and by electron spin resonance spectroscopy of 16- and 5-doxyl stearic acid, respectively. The fluidity of the surface region of the lipid bilayer of the tonoplast vesicles decreased by chilling. The fluidity of the surface region of the liposomes and the proton pumping across the reconstituted proteoliposomes with tonoplast H+-ATPase decreased with increasing content of the glycolipids. The proton pumping across chimera proteoliposomes was reduced by chilling only when it was reconstituted in the presence of tonoplast glycolipids from chilled Boro cells. These data suggest that the reduction in ATP-generated proton pumping across the tonoplast by chilling is due to the decrease in the fluidity of the surface region of the lipid bilayer of the tonoplast, which is caused by the changes in glycolipids.  相似文献   

18.
F Ricchelli  S Gobbo  G Moreno  C Salet 《Biochemistry》1999,38(29):9295-9300
The dynamic properties of protein and lipid regions of mitochondrial membranes during the permeability transition (PT) process were studied by following the anisotropy changes of hematoporphyrin (HP) and 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. We show that opening of the PT pore is accompanied by a remarkable increase of mitochondrial membrane fluidity which is specifically localized to protein sites, while lipid domains are unaffected. The increased membrane fluidity is not related to the collapse of transmembrane potential that follows the PT, as demonstrated by a comparison between the anisotropy properties of permeabilized mitochondria and impermeable, depolarized organelles. Parameters such as osmotic swelling and temperature, which are shown to affect the mitochondrial membrane dynamics in the absence of permeability transition, cannot alone account for the pore dynamical properties. We suggest that the observed increase in fluidity is mainly due to a conformational change of pore-forming protein(s) during the "assembly" of the PT pore.  相似文献   

19.
Solubilization and interaction of α-tocopherol into bis(2-ethylhexyl)sulphosuc cinate sodium salt microemulsion systems have been studied by temperature dependent phase transition, viscosity and nuclear magnetic resonance studies. Tocopherol being an amphiphilic molecule dissolves into the interfacial surfactant monolayer of the microemul sion droplets. The dissolution leads to an enhancement of the rigidity of the surfactant monolayer as studied by the increase in mixing and phase transition temperatures of the microemulsion droplets. Solubilization of tocopherol into microemulsion droplets causes an increase in the effective size of the droplet and as a consequence, the inter-droplet interactions are also increased. The water binding capacity of the surfactant (bis(2-ethylhexyl)sulphosuccinate sodium salt) is reduced due to solubilization of tocopherol as is evidenced from the downfield shifts of water proton magnetic resonances. In the presence of the dissolved electrolytes into the aqueous core, tocopherol is squeezed out of the microemulsion droplets increasing the membrane fluidity and permeability.  相似文献   

20.
Membrane composition and ion-permeability in extremophiles   总被引:1,自引:0,他引:1  
Abstract: Protons and sodium ions are the only used coupling ions in energy transduction in Bacteria and Archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high as compared to sodium ions. In some thermophiles, therefore, sodium is the sole energy coupling ion. Comparison of the proton- and sodium permeability of the membranes of variety of bacterial and archaeal species that differ in their optimal growth temperature reveals that the permeation processes of protons and sodium ions must occur by different mechanisms. The proton permeability increases with the temperature, and has a comparable value for most species at their respective growth temperatures. The sodium permeability is lower than the proton permeability and increases also with the temperature, but is lipid independent. Therefore, it appears that for most bacteria the physical properties of the cytoplasmic membrane are optimised to ensure a low proton permeability at the respective growth temperature.  相似文献   

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