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1.
Cytochrome b5 was extracted and purified from beef liver by a detergent method (cytochrome d-b5). The hydrophilic moiety which carries the heme group (cytochrome t-b5) was prepared by trypsin action upon pure cytochrome d-b5.Single-shelled lecithin liposomes form complexes with cytochromes d-b5 up to a molar ratio of one protein for 35 phospholipids. The lipid-protein complexes were isolated by gel filtration on Sepharose 4B. They are hollow vesicles in which [3H]-glucose can be trapped. Their diameter is greater than that of the initial liposomes.Cytochrome t-b5 does not interact with the vesicles. These results show that the hydrophobic tail is necessary for the binding and that the hydrophilic part of the protein is located on the outer face of the vesicles. This asymmetry is also proved by the action of reducing agents.Experiments with saturated phosphatidylcholines show that the protein interacts with the lipids both below the transition temperature TM. i.e. when the aliphatic chains are in a crystalline state, and above TM, when the alipathic chain are in a fluid state.1H NMR spectra show that even at the maximum cytochrome d-b5 concentration the presence of the proteins does not markedly change the dynamics to the phospholipid molecules. An asymmetric single-shelled vesicle structure is proposed for the complex.  相似文献   

2.
The intact, amphipatic form of cytochrome b5 could bind to unsealed ghosts, but not to resealed ghosts, suggesting that the cytochrome could bind only to the inner (cytoplasmic) surface of the ghost membrane. This was further confirmed by the finding that the cytochrome could bind to closed, inside-out vesicles prepared from the ghosts. This asymmetric binding was not due to the exclusive localization of sialic acid and sugar chains on the outer surface of the ghosts membrane, because the cytochrome could not bind to ghosts even after enzymatic removal of these components. Although liposomes consisting of phosphatidylcholine or both phosphatidylcholine and sphingomyelin could effectively bind the cytochrome, this binding capacity was progressively decreased as increasing amount of cholesterol was included in the composition of phosphatidylcholine liposomes. Removal of cholesterol from resealed ghosts by incubation with egg phosphatidylcholine liposomes resulted in the binding of cytochrome b5 to the outer surface of the treated ghosts. The possibility is discussed that the asymmetric binding is due to preferential localization of cholesterol in the outer leaflet of the lipid bilayer that constitutes the ghost membrane.  相似文献   

3.
Secretory vesicles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca2+. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesicles in freeze-fractured suspensions of vesicles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10?7 M and 10?4 M Ca2+, was specific for Ca2+, was inhibited by other divalent cations and was abolished by pretreatment of vesicles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca2+ in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesicles: Ca2+ could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type II of intact secretory vesicles as well as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca2+ concentrations we have not found a major influence of Ca2+ asymmetry on fusion. Incorporation of the ganglioside GM3, which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca2+-selective electrode we have identified in secretory vesicle membranes both high affinity binding sites for Ca2+ (Kd = 1.6 · 10?6M) and low affinity sites (Kd = 1.2 · 10?4M).  相似文献   

4.
According to previous authors, cytochrome b5, when extracted from bovine liver by a detergent method, is called cytochrome d-b5. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome t-b5.Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, R, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome d-b5, whereas R does not change for cytochrome c and cytochrome t-b5. This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome d-b5 occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome d-b5, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.  相似文献   

5.
Dipalmitoylphosphatidylcholine (DPPC) dispersed in perdeuterated glycerol was investigated in order to determine the effects on the Raman spectra of hydrocarbon chain interdigitation in gel-phase lipid bilayers. Interdigitated DPPC bilayers formed from glycerol dispersions in the gel phase showed a decrease in the peak height intensity I2850/I2880 ratio, for the symmetric and asymmetric methylene CH stretching modes, respectively, as compared to non-interdigitated DPPC/water gel-phase dispersions. The decrease in this spectral ratio is interpreted as an increase in chain-chain lateral interactions. Spectra recorded in the 700–740 cm?1 CN stretching mode region, the 1000–1200 cm?1 CC stretching mode region and the 1700–1800 cm? CO stretching mode region were identical for both the interdigitated and non-interdigitated hydrocarbon chain systems. At low temperatures the Raman peak height intensity ratios I2935/I2880 were identical for the DPPC/glycerol and DPPC/water dispersions, indicating that this specific index for monitoring bilayer behavior is insensitive to acyl chain interdigitation. The increase, however, in the change of this index at the gel-liquid crystalline phase transition temperature for the DPPC/glycerol dispersions implies a larger entropy of transition in comparison to the non-interdigitated DPPC/water bilayer system.  相似文献   

6.
The dielectric dispersion in the MHz range of the zwitterionic dipolar phosphocholine head groups has been measured from 0–70°C for various mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. The abrupt change in the derived relaxation frequency f2 observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42°C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42°C no sudden changes in f2 are found at 20 or 33 mol% cholesterol, where phase boundaries have been suggested from other methods. Above 42°C, however, a decrease in f2 at cholesterol contents up to 20–30 mol% is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules.  相似文献   

7.
Purified cytochrome P450SCC from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of P450SCC into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When P450SCC was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the P450SCC-containing liposomes showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, P450SCC was less stable than P450SCC in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal P450SCC. Liposomal P450SCC required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal P450SCC was subjected to p-chloromercuriphenyl sulfonic acid treatment. This reagent destroyed the liposomal P450SCC. These results suggest that the heme is located in the proximity of the p-chloromercuriphenyl sulfonic acid reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.  相似文献   

8.
The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of 125I-labeled toxin B-IV by native toxin B-IV have shown specific binding of 125I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [3H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association (k+1=7.3·105M?1·s?1) and dissociation (k? 1=2·10?3s?1) shows a nearly identical Kd of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.  相似文献   

9.
Cationic and uncharged forms of a tertiary amine local anesthetic are reported to have different properties and potencies as nerve blocking agents. However, the relative capacities of each form of the local anesthetic to perturb the properties of different model membrane systems is unknown. For this reason we have studied the effects of uncharged lidocaine (high pH) and its quaternary amine analogue (W49091) on the phase transition properties of DMPS, DPPE and DPPC liposomes using high-sensitivity differential scanning calorimetry. We report that neutral lidocaine interacts similarly with all three phospholipids. This interaction results in a decrease in the temperature of the gel å liquid crystalline phase transition (Tm), an increase in the enthalpy of the transition (ΔH), and a slight decrease in the cooperativity of melting. Quaternary lidocaine (W49091), on the other hand, interacts significantly with only DMPS; the result being again a decrease in the temperature of DMPS melting, an increase in ΔH, and a slight decrease in the cooperativity of the phase transition. These results are interpreted to indicate that uncharged lidocaine enters the membrane during the DPPE and DPPC phase transitions. In the case of DMPS, an influx of both charged forms of lidocaine must occur at Tm. These anesthetic fluxes at the lipid's phase transition are suggested to be responsible for the observed elevated enthalpies of the respective transitions. The observation that the cationic form of lidocaine does not significantly modify the behavior of DPPC and DPPE liposomes suggests that these lipids are not important components of the anesthetic's site in nerve membranes. However, the dramatic perturbation of the properties of DMPS by W49091 suggests that phosphatidylserine may comprise part of this inhibitory site.  相似文献   

10.
Glycerol substitutes for water in multilamellar phosphatidylcholine liposomes in that the fluid spaces between bilayers, as well as their main transition temperatures, heat capacities, and ethalpies are very similar in water and in pure glycerol. One major difference is that the gel state phase of dipalmitoylphosphatidylcholine (DPPC) in glycerol consists of bilayers with fully interdigitated hydrocarbon chains. Interdigitated DPPC phases are also formed in ethylene glycol or in methanol (at low methanol content). In solutions of glycerol and water, the fluid spacing between bilayers is a function of mole fraction of glycerol Xg, reaching maximum values at Xg ≌ 0.1 for lipid in the liquid crystalline phase and at Xg ≌ 0.3 for the gel phase. These changes are explained in terms of a modification of the long-range Van der Waals attractive forces by glycerol.  相似文献   

11.
125I-labelled α2-macroglobulin complexed with thrombin or plasmin bound to hepatocytes in a concentration-and time-dependent manner. The apparent Kd values calculated from displacement experiments were 7.9 · 10?8 M for α2-macroglobulin-thrombin and 8.5 · 10?8 M for α2-macroglobulin-plasmin. Association of these complexes was only partially reversible; after a 180 min incubation period, 50–60% of the bound radioactivity was internalized by the cells. α2-Macroglobulin itself bound also to hepatocytes, but the affinity of the α2-macroglobulin complexes was higher than that of the inhibitor alone, and α2-macroglobulin was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their α2-macroglobulin complexes. The α2-macroglobulin-thrombin complex competed with the α2-macroglobulin-plasmin complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for α2-macroglobulin-proteinase and antithrombin III-proteinase complexes are different.  相似文献   

12.
In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [3H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu5AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg / ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times (t12) for at least one-third of the cell cholesterol of 3.2 ± 0.6 and 14.3 ± 1.5 h, respectively. Plasma membrane vesicles (0.5–5.0 μm diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the t12 values for plasma membrane vesicles of Fu5AH and WIRL cells are 3.9 ± 0.5 and 11.2 ± 0.7 h, respectively. These t12 values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rate indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu5AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu5AH and WIRL cells were the same, with values of 3.1 ± 0.1 and 2.9 ± 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in t12 values for cholesterol efflux from these cell lines.  相似文献   

13.
Perturbations induced by melittin on the thermotropism of dimyristoyl-, dipalmitoyl-, distearoylphosphatidylcholine and natural sphingomyelin are investigated and rationalized from data obtained by fluorescence polarization, differential scanning calorimetry and Raman spectroscopy. Depending on the technique and / or experimental conditions used, the observed effects differ at the same lipid to protein molar ratio, due to partial binding of melittin. The binding is more efficient for tetrameric than for monomeric melittin, but in both cases its affinity is weaker for phosphatidylcholine dispersions in the gel phase than for sonicated vesicles. For temperatures T ? Tm efficient binding occurs whatever the initial state of the lipids is. One can summarize the effects induced by melittin on the transition temperature as follows: (i) No upward shift is observed on synthetic phosphatidylcholines when lipid degradation is avoided. This is achieved by using highly purified melittin, phospholipase inhibitors, and / or non-hydrolysable lipids. (ii) Melittin monomer does not change Tm. (iii) When melittin tetramer is stabilized, it decreases Tm by 10–15 deg. C. The transition broadens, and is finally abolished for Ri ? 2. Very similar results are found for natural sphingomyelin. Fluorescence polarization indicates similar changes in order and dynamics of the acyl chains for all lipid studied. For T ? Tm, fluorescence and Raman show that melittin decreases the amount of CH2 groups in ‘trans’ conformation and the intermolecular order of the chains. According to fluorescence data, there is an increase of the rigid-body orientational order at T ? Tm, while from Raman the positional intermolecular order decreases without significant change in the CH2 groups ‘trans’/‘gauche’ ratio.  相似文献   

14.
Differential polarized phase fluorometry was used to quantify the rotational rate (R) and limiting anisotropy (r) of the membrane probe diphenylhexatriene (DPH) in solvents and lipid vesicles exposed to hydrostatic pressures ranging from 1 bar to 2 kbar. These measurements reveal the effect of pressure on the phase-transition temperatures of the phosphatidylcholine vesicles, and the effects of pressure on order parameter of the acyl side-chain region of the membranes, the latter as indicated by r. In addition to the well-known elevation of the transition temperature (Tc) with pressure, our results demonstrate that increased pressure restores the order of the bilayers to that representative of temperatures below the transition temperature. We also found that solvents which allowed free isotropic rotation of DPH at 1 bar no longer allowed free rotation when sufficiently compressed; moreover, the apparent DPH rotational rate increased with r. Pressure studies using both DPH and the charged DPH analogue, trimethylammonium DPH (TMA-DPH) indicated that the Tc of dipalmitoylphosphatidylcholine vesicles increased 23 K/kbar and an apparent volume change of 0.036 ml/mol lipid at the phase transition. Assuming, as has been proposed, that TMA-DPH is localized near the glycerol backbone region of the bilayers, these results indicate a similar temperature- and pressure-dependent phase transition in this region and the acyl side-chain region of the membrane.  相似文献   

15.
Several characteristics of calmodulin association with brain synaptic and coated vesicles were analyzed and compared. Radioimmunoassay revealed that both classes of vesicles contain approx. 1 μg of calmodulin per mg of vesicle protein. Discontinuous sucrose gradients revealed that coated and synaptic vesicles preparations were homogeneous and had different sedimentation properties. Binding of 125I-labeled calmodulin to synaptic and coated vesicles was Ca2+ dependent and displaced by unlabeled calmodulin but not by troponin-C. Scatchard analysis revealed the presence of two binding sites. In both vesicle types there was one high-affinity, low-binding-capacity site (Kd = 1–39 nM and Bmax = 4–16 pmol/mg) and one low-affinity, high-binding-capacity site (Kd = 102–177 nM and Bmax = 151–202 pmol/mg). (Ca2+ + Mg2+)-ATPase activity was stimulated in both synaptic and coated vesicles by calmodulin. Thus synaptic and coated vesicles may possess similar calmodulin binding sites.  相似文献   

16.
The phase transition temperature (Tt) of dipalmitoyl phosphatidic acid multilamellar liposomes is depressed 10°C by the inhalation anesthetic methoxyflurane at a concentration of 100 mmol/mol lipid. Application of 100 atm of helium pressure to pure phosphatidic acid liposomes increased Tt only 1.5°C. However, application of 100 atm helium pressure to dipalmitoyl phosphatidic acid lipsomes containing 100 mmol methoxyflurane/mol lipid almost completely antagonized the effect of the anesthetic. A nonlinear pressure effect is observed. In a previous study, a concentration of 60 mmol methoxyflurane/mol dipalmitoyl phosphatidylcholine depressed Tt only 1.5°C, exhibiting a linear pressure effect. The completely different behavior in the charged membrane is best explained by extrusion of the anesthetic from the lipid phase.  相似文献   

17.
The protein-induced lipid transfer between phosphatidylcholine vesicles was investigated. Measurements of the degree of polarization at single vesicles were made by flow cytometry using diphenylhexatriene as the optical probe. Vesicles differing in phase transition temperature could be distinguished by their degree of polarization at a temperature where one population was in the fluid (T > Tt) and the other one in the quasi-crystalline (T < Tt) state. Besides vesicles containing exchanged lipids we also observed fractions of unaffected vesicles. The lipid exchange was visualized directly by freeze-fracture electron microscopy. The characteristic ‘ripple’ structure of phosphatidylcholine vesicles disappeared upon exchange with lipid in the fluid state.  相似文献   

18.
Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous n-alcohols from methanol to n-heptanol caused an inhibition of calcium uptake and an enhancement of ATPase activity. The n-alcohol treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of n-alcohol-treated vesicles was about twice that of control vesicles. With increasing number (n) of carbon atoms of the n-alcohols, the maximum increment of ATPase activity increased, and both the alcohol concentration (NCa) required to inhibit calcium uptake by 50% and the alcohol concentration (NATPase) required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows.
NCa=23.5·10?0.593nM
NATPase=35.5·10?0.593nM
The ratio, NATPase to NCa, was constant for all n values. The apparent free energy of binding of the methylene groups of n-alcohols to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of n-alcohols in octanol and water (?670 cal/mole). The effects of n-alcohols on membrane vesicles are discussed on the basis of these data.  相似文献   

19.
Band 3 protein, extracted from human erythrocyte membranes by Triton X-100, was recombined with egg lecithin/cholesterol mixtures to form small unilamellar vesicles at a yield of 15–20%. These systems exhibited sulfate fluxes which were inhibitable by stilbene disulfonates and other inhibitors. Maximal inhibition could only be obtained when inhibitors were present at both membrane surfaces. Inhibitor constants I50 were higher than in the native membrane. Quantitatively, transport function was retained at least 60%, as related to the amount of protein involved. Sulfate transport in the recombinates resembled transport in the native membrane with respect to temperature dependence (Ea = 29?32 kcal/mol), pH dependence between pH 6.5 and 7.8, and the relationship between net and exchange fluxes. In contrast to the native cell, concentration dependence was linear up to 80 mM sulfate, which may be indicative of a lowered affinity for the substrate. Lactate transport in these systems, although substantial, was insensitive to stilbene disulfonates as well as to mercurials, indicating that band 3 is not involved in the specific monocarboxylate transfer in the erythrocyte. Anion transport in band 3-lipid recombinates was insensitive to cholesterol between 0 and 27 mol%. Treatment with proteases, while not affecting transport per se, abolished sensitivity to stilbene disulfonate inhibitors. These observations indicate a number of disturbances of band 3 after recombination, in spite of a preservation of the major transport properties.  相似文献   

20.
The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant (K′s) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the Ks and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome P-450 is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome P-450 binding reaction and in the temperature dependency of the Ks.  相似文献   

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