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Microinjection in combination with microfluorimetry to study proton diffusion along phospholipid membranes 总被引:1,自引:0,他引:1
Proton diffusion along the surface of a planar bilayer lipid membrane was measured by means of acid/base injection with a micropipette and recording of the kinetics of fluorescence changes of fluorescein-labelled lipid on the surface. The dimensionality of the process was assayed by fitting the kinetic curves with two-dimensional (2D) or three-dimensional (3D) diffusion equations. In agreement with Serowy et al. (Biophys J 84:1031-1037, 2003), lateral proton diffusion proceeded via bulk phase by means of buffer molecules as proton carriers (D = 600 microm2/s) under the conditions of 1 mM buffer in the solution. Introduction of proton binding sites on the membrane surface led to the appearance of a considerable contribution of two-dimensional proton diffusion on the membrane surface with D = 1,100 mum(2)/s. The system described can be used to study the dependence of the proton diffusion rate on the phospholipid and protein composition of the membrane. 相似文献
3.
The lateral diffusion coefficient of ganglioside GM1 incorporated into preformed dimyristoylphosphatidylcholine (DMPC) vesicles has been investigated under a variety of conditions using the technique of fluorescence photobleaching recovery. For these studies the fluorescent probe 5-(((2-Carbohydrazino)methyl)thio)acetyl) amino eosin was covalently attached to the periodate-oxidized sialic acid residue of ganglioside GM1. This labeled ganglioside exhibited a behavior similar to that of the intact ganglioside, and was able to bind cholera toxin. The lateral diffusion coefficient of the ganglioside was dependent upon the gel-liquid crystalline transition of DMPC. Above Tm the lateral diffusion coefficient of the ganglioside was 4.7 X 10(-9) cm2 s-1 (with greater than 80% fluorescence recovery). This diffusion coefficient is significantly slower than the one previously observed for phospholipids in DMPC bilayers. The addition of increasing amounts of ganglioside, up to a maximum of 10 mol %, did not have a significant effect on the lateral diffusion coefficient or in the percent recovery. At 30 degrees C, the lateral mobility of ganglioside GM1 was not affected by the presence of 5 mM Ca2+, suggesting that, at least above Tm, Ca2+ does not induce a major perturbation in the lateral organization of the ganglioside molecules. The addition of stoichiometric amounts of cholera toxin to samples containing either 1 or 10 mol % ganglioside GM1 produced only a small decrease in the measured diffusion coefficient. The fluorescence recovery after photobleaching experiments were complemented with excimer formation experiments using pyrene-phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Membrane fusion. Transfer of phospholipid molecules between phospholipid bilayer membranes 总被引:2,自引:0,他引:2
Fusion of phosphatidylcholine (PC) vesicles and of PC-phosphatidylserine (PS) vesicles has been studied using spin-labeled PC and PS. Analysis of ESR spectra indicated transfer of phospholipid molecules between phospholipid vesicles at the instant of membrane contact by vesicular collision. The transfer rate of PC was not greatly affected by the presence of the anionic lipid in the membranes. The rate of PC transfer between PS-PC vesicles was nearly the same as that of PS transfer. Calcium ion greatly enhanced the transfer of phospholipid molecules between PS-PC vesicles. The enhancement of PS transfer occurred instantaneously. The phospholipid transfer is related to the fusion of vesicles. 相似文献
5.
L Thilo 《Biochimica et biophysica acta》1977,469(3):326-334
In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469,311--325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, k--, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are kP- = (0.86 +/- 0.05) - 10(-5) S-1 and ke- = (1.09 +/- 0.13) - 10(-6) s-1 for phospholipid molecules with trans-delta 9-hexadecenoate and trans-delta 9-octadecenoate, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids. 相似文献
6.
Dynamic properties of gramicidin A in phospholipid membranes 总被引:3,自引:0,他引:3
The flexibility of the tryptophan side chains of gramicidin A and the rotational diffusion of the peptide in methanolic solution and in three membrane systems were studied with deuterium nuclear magnetic resonance (NMR). Gramicidin A was selectively deuterated at the aromatic ring systems of its four tryptophan side chains. In methanolic solution, the tryptophan residues remained immobile and served as a probe for the overall rotation of the peptide. The experimentally determined rotational correlation time of tau c = 0.6 X 10(-9) s was consistent with the formation of gramicidin A dimers. For gramicidin A incorporated into bilayer membranes, quite different results were obtained depending on the chemical and physical nature of the lipids employed. When mixed with 1-palmitoyl-sn-glycero-3-phosphocholine (LPPC) at a stoichiometric lipid:peptide ratio of 4:1, gramicidin A induced the formation of stable bilayer membranes in which the lipids were highly fluid. In contrast, the gramicidin A molecules of this membrane remained completely static over a large temperature interval, suggesting strong protein-protein interactions. The peptide molecules appeared to form a rigid two-dimensional lattice in which the interstitial spaces were filled with fluidlike lipids. When gramicidin A was incorporated into bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) above the lipid phase transition, the deuterium NMR spectra were motionally narrowed, indicating large-amplitude rotational fluctuations. From the measurement of the quadrupole echo relaxation time, a rotational correlation time of 2 X 10(-7) s was estimated, leading to a membrane viscosity of 1-2 P if the rotational unit was assumed to be a gramicidin A dimer. (ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
《BBA》2013,1827(10):1165-1173
Proton matrix ENDOR was performed to investigate the protons close to the manganese cluster in oriented samples of photosystem II (PS II). Eight pairs of ENDOR signals were detected in oriented PS II membranes. At an angle of θ = 0° between the membrane normal vector n and the external field H0, five pairs of ENDOR signals were exchangeable in D2O medium and three pairs were not exchangeable in D2O medium. The hyperfine splitting of 3.60 MHz at θ = 0° increased to 3.80 MHz at θ = 90°. The non-exchangeable signals with 1.73 MHz hyperfine splitting at θ = 0°, which were assigned to a proton in an amino acid residue, were not detected at θ = 90° in oriented PS II or in non-oriented PS II. Highly resolved spectra show that only limited numbers of protons were detected by CW-ENDOR spectra, although many protons were located near the CaMn4O5 cluster. The detected exchangeable protons were proposed to arise from the protons belonging to the water molecules, labeled W1-W4 in the 1.9 Å crystal structure, directly ligated to the CaMn4O5 cluster, and nearby amino-acid residue. 相似文献
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Rotating-frame relaxation studies of slow motions in fluorinated phospholipid model membranes. 总被引:5,自引:3,他引:2
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Rotating-frame relaxation experiments have been carried out on 19F-labeled dimyristoylphosphatidylcholine model membranes. The lipids are labeled with a single CF2 group in the 4-, 8-, or 12-position of the 2-acyl chain. Both oriented lipid bilayers and multilamellar liposomes have been investigated. The relaxation rate has been measured as a function of the locking-field strength, the sample orientation, the label position, and the temperature. Our results have confirmed that extensive slow motions exist in the bilayer and dominate the low-frequency relaxation. The relaxation rate is quite sensitive to the label position. However, many other features of the relaxation are very similar for all three lipid isomers. The temperature dependence of the relaxation rate for the multilamellar liposomes differs from the oriented bilayers, which may imply that the motions are also different. To fit our data, a working model consisting of a superposition of an anisotropic reorientation term and a director fluctuation term has been proposed. We have also verified that almost all of the relaxation process is caused by modulations of the intramolecular interactions. Based on this, a view of the slow motions at a molecular level is discussed in this paper. 相似文献
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T. R. Hennessy 《Bulletin of mathematical biology》1971,33(2):249-257
In this paper, a tractable mathematical model is proposed to describe transient inert gas diffusion in heterogeneous tissue
with perfusion controlling gas input to the cellular region. The corresponding solution of overall mass uptake of the inert
gas is derived exactly and should be useful in interpreting washout curves from particular tissue zones, whether there is
any interaction with cellular diffusion or not. It is shown that the solution contains effectively nearly all models hitherto
proposed to describe gas uptake in tissue. However some indication is given of a possible situation where perfusion, extra-cellular
and cellular diffusion will need to be treated separately. 相似文献
11.
Klaas J. Hellingwerf 《Journal of bioenergetics and biomembranes》1987,19(3):225-238
Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochromec and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ0), a lipid-and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ0H2) and oxidized cytochromec. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochromec and UQ0. At 250 and 25 µM UQ0 and cytochromec, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.Abbreviations RC
reaction center
- UQ0/UQ0H2
oxidized and reduced form of 2,3-dimethoxy-5-methyl-1,4-benzoquinone
- D/D+
reduced and oxidized form of the primary electron donor of the RCs
- CCCP
carbonylcyanide-trichloromethoxy phenylhydrazone
- UQA/UQ
A
–
oxidized and semiquinone form of the primary electron acceptor of the RCs
- UQB/UQ
B
–
/UQBH2
oxidized, semiquinone, and reduced form of the secondary electron acceptor of the RCs
- LDAO
lauryldimethylamine-N-oxide
During the course of this study K.J.H. was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). This research was supported by grants from the National Institutes of Health (EY-02084) and from the Office of Naval Research (ONR-NOOO 14-79-C 0798) to M. Montal. 相似文献
12.
Michael Singer 《Chemistry and physics of lipids》1979,25(1):15-28
The passage of a phospholipid through the gel to liquid crystal phase transition is associated with an increase in the motional freedom of its fatty acyl chains as measured by spectroscopic techniques and an essentially isothermal absorption of heat as measured by differential scanning calorimetry (DSC). In addition, bilayers formed from that phospholipid display a permeability maximum for both non-electrolytes and electrolytes in the temperature region of the phase transition. In this study the sodium (and in some cases glucose) permeabilities of liposomes composed of either dimyristoyl or dipalmitoyl phosphatidylcholine plus dicetylphosphate were measured in the presence of a group of benzene and adamantane derivatives known to increase fatty acyl chain motion below the lipid transition temperature (Tc) and in the case of the adamantanes to also lower the Tc as measured by DSC. None of these compounds change the temperature at which the permeability maximum occurs despite their lowering of the phospholipid Tc. That is, in the presence of these additives there is observed an apparent dissociation between the phase transition and the permeability maximum. It is proposed that the permeability maximum normally observed in the temperature region of the Tc is associated with the completion of the ‘melting’ process. Hence a compound could cause early ‘melting’ of the bilayer but not change its permeability properties if the temperature at which the ‘melting’ process neared completion was not changed. 相似文献
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The action of certain antibiotics on mitochondrial, erythrocyte and artificial phospholipid membranes. The role of induced proton permeability 总被引:39,自引:21,他引:18
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1. The action of the antibiotics enniatin A, valinomycin, the actin homologues, gramicidin, nigericin and dianemycin on mitochondria, erythrocytes and smectic mesophases of lecithin–dicetyl hydrogen phosphate was studied. 2. These antibiotics induced permeability to alkali-metal cations on all three membrane systems. 3. The ion specificity on each membrane system was the same. 4. Enniatin A, valinomycin and the actins did not induce permeability to protons, whereas nigericin and dianemycin rendered all three membrane systems freely permeable to protons. 5. Several differences were noted between permeability induced by nigericin and that induced by gramicidin. 6. The action of all these antibiotics on mitochondrial respiration could be accounted for by changes in passive ion permeability of the mitochondrial membrane similar to those induced in erythrocytes and phospholipid membranes, if it is assumed that a membrane potential is present in respiring mitochondria. 相似文献
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Proton NMR spectroscopy was used to demonstrate that transmembrane pH gradients across single-bilayer vesicle membranes effect the transport and concentration of carboxylic acids. The results obtained indicate that this transport occurs selective permeation of the membrane by the protonated (uncharged) form of the acid. 相似文献
17.
Intrinsic electric fields and proton diffusion in immobilized protein membranes. Effects of electrolytes and buffers.
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We present a theory for proton diffusion through an immobilized protein membrane perfused with an electrolyte and a buffer. Using a Nernst-Planck equation for each species and assuming local charge neutrality, we obtain two coupled nonlinear diffusion equations with new diffusion coefficients dependent on the concentration of all species, the diffusion constants or mobilities of the buffers and salts, the pH-derivative of the titration curves of the mobile buffer and the immobilized protein, and the derivative with respect to ionic strength of the protein titration curve. Transient time scales are locally pH-dependent because of protonation-deprotonation reactions with the fixed protein and are ionic strength-dependent because salts provide charge carriers to shield internal electric fields. Intrinsic electric fields arise proportional to the gradient of an "effective" charge concentration. The field may reverse locally if buffer concentrations are large (greater to or equal to 0.1 M) and if the diffusivity of the electrolyte species is sufficiently small. The "ideal" electrolyte case (where each species has the same diffusivity) reduces to a simple form. We apply these theoretical considerations to membranes composed of papain and bovine serum albumin (BSA) and show that intrinsic electric fields greatly enhance the mobility of protons when the ionic strength of the salts is smaller than 0.1 M. These results are consistent with experiments where pH changes are observed to depend strongly on buffer, salt, and proton concentrations in baths adjacent to the membranes. 相似文献
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The effects of photosystem II (PSII) exogenous electron donors and acceptors on the kinetics of weak light photoinhibition of NH2OH/EDTA-extracted spinach PSII membranes were examined. Under aerobic conditions, Mn2+ (approximately 1 Mn/reaction center; Km approximately 400 nM) inhibited photoinactivation and approximately 1 Mn/reaction center plus 100 microM NH2NH2 gave almost complete protection. In the absence of electron donors, strict anaerobiosis greatly inhibited photoinactivation even in the presence of an electron acceptor. Under aerobic conditions, the addition of electron acceptors (FeCN, DCIP), oxyradical scavengers, or superoxide dismutase strongly suppressed rates of photodamages. Increase in the concentrations of superoxide above those produced by illuminated NH2OH/EDTA-photosystem II membranes increased the rates of damage in the light but gave no damage in the dark. Scavengers of hydroxyl radicals and singlet oxygen did not suppress the rates of aerobic photoinhibition. These findings, along with others, lead us to conclude that photodamage of the secondary donors of the PSII reaction center occurs by two mechanisms: (1) a rapid superoxide and tyrosine YZ+ dependent process and (2) a slower process in which P680+/Chl+ catalyze the damages. 相似文献
20.
Effect of cholesterol, divalent ions and pH on spherical bilayer membrane fusion was studied as a function of increasing temperature. Spherical bilayer membranes were composed of natural [phosphatidylcholine (PC) and phosphatidylserine (PS)] as well as synthetic (dipalmitoyl-PC, dimyristoyl-PC and dioleoyl-PC) phospholipids. Incorporation of cholesterol into the membrane (33% by weight) suppressed the fusion temperature and also greatly reduced the percentage of membrane fusion. The presence of 1 mM divalent ions (Ca++, Mg++ or Mn++) on both sides or one side of the PC membrane did not affect appreciably its fusion characteristic with temperature, but the PS membrane fusion with temperature was greatly enhanced by the presence of divalent ions. The variation of pH of the environmental solution in the range of 5.5 approximately 7.0 did not affect the membrane fusion characteristic. However, at pH 8.5, the fusion with respect to temperature was shifted toward the lower temperature by approximately 3degreesC for PC and PS membranes, and at pH 3.0 the opposite situation was observed as the fusion temperature was increased by 6degreesC for PS membranes and by 4degreesC for PC membranes The results seem to indicate that membrane fluidity and structural instability in the bilayer are important for membrane fusion to occur. 相似文献