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1.
Sonication of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC, up to approximately 30 mol %) produces small unilamellar vesicles (SUV, 250-265 A diameter). Phosphorus-31 NMR of the POPC/lysoPC vesicles gives rise to four distinct peaks for POPC and lysoPC in the outer and in the inner bilayer leaflet which can be used to localize and quantify the phospholipids in both vesicle shells. Addition of paramagnetic ions (3 mM Pr3+) enhances outside/inside chemical shift differences and allows monitoring of membrane integrity by the absence of Pr3+ in the vesicle interior. 31P NMR shows that lysoPC in these highly curved POPC/lysoPC vesicles prefers the outer bilayer leaflet. LysoPC incorporation into POPC SUV furthermore causes a substantial and concentration-dependent decrease in spin-spin relaxations (T*2) of the outside POPC phosphorus signals from 55 ms for pure POPC vesicles (v1/2, 5.8 Hz) to 29.5 ms (v1/2, 10.8 Hz) for POPC/lysoPC vesicles containing 25 mol % lysoPC. Our findings are consistent with the idea of a cone-shaped lysoPC molecule which, for geometric reasons, is preferentially accommodated in the outer bilayer leaflet. LysoPC incorporation into POPC SUV restricts POPC headgroup motion and tightens phospholipid packing, but only in the outer bilayer shell.  相似文献   

2.
New vehicles for the non-invasive delivery of agents are introduced. These carriers can transport pharmacological agents, including large polypeptides, through the permeability barriers, such as the intact skin. This capability depends on the self-regulating carrier deformability which exceeds that of the related but not optimized lipid aggregates by several orders of magnitude. Conventional lipid suspensions, such as standard liposomes or mixed lipid micelles, do not mediate a systemic biological effect upon epicutaneous applications. In contrast to this, the properly devised adaptable carriers, when administered on the intact skin, transport therapeutic amounts of biogenic molecules into the body. This process can be nearly as efficient as an injection needle, as seen from the results of experiments in mice and humans with the insulin-carrying vesicles. The carrier-mediated transcutaneous insulin delivery is unlikely to involve shunts, lesions or other types of skin damage. Rather than this, insulin is inferred to be transported into the body between the intact skin cells with a bio-efficiency of at least 50% of the s.c. dose action.  相似文献   

3.
This study investigated the biodistribution of bovine brain sphingomyelin (SM)/cholesterol (CH) (2/1; M/M) small unilamellar vesicles (SUV) in mice, addressing specifically the volume of distribution and transcapillary passage of the SUV. The complex of nitrilotriacetic acid with In-111 or Ga-67 ions was encapsulated in the SUV as the radioactive marker for various studies. The structural integrity of liposomes in vitro and in vivo was monitored by the technique of gamma ray perturbed angular correlation. Our data suggested that initially the SM/CH SUV remained within the vascular system and occupied a volume of distribution approximately 1.28 times larger than that of erythrocytes in the vascular system of mice. However, our data also indicated that with time the SM/CH SUV could get out of the vascular system of mice and were taken up by surrounding tissues over a period of 24 hours.  相似文献   

4.
Giant unilamellar vesicles (GUVs) are simple model membrane systems of cell-size, which are instrumental to study the function of more complex biological membranes involving heterogeneities in lipid composition, shape, mechanical properties, and chemical properties. We have devised a method that makes it possible to prepare a uniform sample of ternary GUVs of a prescribed composition and heterogeneity by mixing different populations of small unilamellar vesicles (SUVs). The validity of the protocol has been demonstrated by applying it to ternary lipid mixture of DOPC, DPPC, and cholesterol by mixing small unilamellar vesicles (SUVs) of two different populations and with different lipid compositions. The compositional homogeneity among GUVs resulting from SUV mixing is quantified by measuring the area fraction of the liquid ordered–liquid disordered phases in giant vesicles and is found to be comparable to that in GUVs of the prescribed composition produced from hydration of dried lipids mixed in organic solvent. Our method opens up the possibility to quickly increase and manipulate the complexity of GUV membranes in a controlled manner at physiological buffer and temperature conditions. The new protocol will permit quantitative biophysical studies of a whole new class of well-defined model membrane systems of a complexity that resembles biological membranes with rafts.  相似文献   

5.
W C Wimley  T E Thompson 《Biochemistry》1990,29(5):1296-1303
The rate and extent of spontaneous exchange of dimyristoylphosphatidylcholine (DMPC) from large unilamellar vesicles (LUV) composed of either DMPC or mixtures of DMPC/distearoylphosphatidylcholine (DSPC) have been examined under equilibrium conditions. The phase state of the vesicles ranged from all-liquid-crystalline through mixed gel/liquid-crystalline to all-gel. The exchange rate of DMPC between liquid-crystalline DMPC LUV, measured between 25 and 55 degrees C, was found to have an Arrhenius activation energy of 24.9 +/- 1.4 kcal/mol. This activation energy and the exchange rates are very similar to those obtained for the exchange of DMPC between DMPC small unilamellar vesicles (SUV). The extent of exchange of DMPC in LUV was found to be approximately 90%. This is in direct contrast to the situation in DMPC SUV where only the lipid in the outer monolayer is available for exchange. Thus, transbilayer movement (flip-flop) is substantially faster in liquid-crystalline DMPC LUV than in SUV. Desorption from gel-phase LUV has a much lower rate than gel-phase SUV with an activation energy of 31.7 +/- 3.7 kcal/mol compared to 11.5 +/- 2 kcal/mol reported for SUV. A defect-mediated exchange in gel-phase SUV, which is not the major pathway for exchange in LUV, is proposed on the basis of the thermodynamic parameters of the activation process. Surprisingly, the rates of DMPC exchange between DMPC/DSPC two-component LUV, measured over a wide range of compositions and temperatures, were found to exhibit very little dependence on the composition or phase configuration of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

6.
Gradients across the outer skin layers may result in fields enforcing a lipid flow into or through the intact skin surface provided that lipids are applied in the form of special vesicles. The osmotic gradient, for example, which is created by the difference in the total water concentrations between the skin surface and the skin interior, provides one possible source of such driving force. It is sufficiently strong to push at least 0.5 mg of lipids per hour and cm2 through the skin permeability barrier in the region of stratum corneum. The lipid concentration gradient, on the contrary, does not contribute much to the lipid penetration into dermis. Occlusion, therefore, is detrimental for the vesicle penetration into intact skin.  相似文献   

7.
We have investigated the behavior of two populations of doxorubicin (DXR)-containing phospholipid vesicles with regard to various physical and pharmacological parameters. DXR-containing liposomes were prepared by ultrasonic irradiation, the lipid composition being phosphatidylglycerol (or phosphatidylserine), phosphatidylcholine and cholesterol. The vesicles were fractionated into oligolamellar vesicles (OLV) and small unilamellar vesicles (SUV) by preparative differential ultracentrifugation (150,000 x g for 1 h). Unentrapped DXR was removed by gel exclusion chromatography. OLV and SUV liposomes differed in size (mean diameters, 247 +/- 113 nm and 61 +/- 16 nm, respectively) and number of lamellae (two for OLV, one for SUV). Drug entrapment per unit of lipid was three to 5-fold higher in OLV than in SUV. In both liposome populations more than 95% of the entrapped drug was membrane-associated. Physical studies on these two vesicle populations revealed higher motional restriction and greater susceptibility to iodide-mediated fluorescence collisional quenching of DXR in the small vesicles. OLV showed superior stability in the presence of plasma as determined by the fraction of DXR retained by the vesicles. It was also found that the tissue distribution of DXR in SUV follows a pattern different from that of DXR in OLV and resembling that of soluble DXR. In accordance with these differences in patterns of tissue distribution, animal studies demonstrated that DXR in OLV is significantly less toxic than DXR in SUV and more effective in a tumor model with predominant involvement of the liver. These results indicate that vesicle size and/or number of lamellae play an important role in optimizing liposome-mediated delivery of DXR, and that oligolamellar liposomes are distinctively superior to small unilamellar liposomes when fluid phase formulations (Tm less than 37 degrees C) with bilayer-associated DXR are considered.  相似文献   

8.
Two structurally distinct lipid vesicles are present in pea and bean cotyledons during the first few days of germination. Both were isolated by sucrose density gradient centrifugation without significant morphological changes. Lipid vesicles of one type were elongated into a sausage-like or flattened-saccular shape, and were interassociated into sheets which were usually one vesicle thick. These sheets remained intact during homogenization and centrifugation, because some of the lipid vesicles in the sheet were interconnected through their bounding membranes, and because there seemed to be a bonding substance between adjacent vesicles. These vesicles were called "composite" lipid vesicles to distinguish them from the more usual, or "simple," lipid vesicles of other plant and animal tissues. Lipid vesicles of the other type were usually larger than the composite lipid vesicles and were always spherical in form. These vesicles remained single and did not interassociate into sheets. They were probably equivalent to the simple lipid vesicles of other tissues.  相似文献   

9.
P Raicu  F Mixich 《Mutation research》1992,283(3):215-219
Lipid vesicles (liposomes) have been shown to be a useful vehicle for the delivery of a variety of compounds to cultured cells. Using multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) we were able to deliver the classical mutagen, sodium azide, into human heteroploid HEp-2 cells. With this method sodium azide is not diluted in culture medium, but it is 'focused' into cells, producing chromosomal aberrations and other major genetic damages. Our results indicate that liposomes are suitable vectors for introducing clastogenic substances into cultured human cells.  相似文献   

10.
The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

11.
M E Haque  A J McCoy  J Glenn  J Lee  B R Lentz 《Biochemistry》2001,40(47):14243-14251
The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.  相似文献   

12.
Distribution of 5-doxylstearic acid in the membranes of mammalian cells   总被引:1,自引:0,他引:1  
Concentration-dependent spin broadening of ESR spectra of the nitroxide 5-doxylstearic acid has been used to evaluate the distribution of 5-doxylstearic acid in the membranes of intact mouse thymus-bone marrow (TB) and Chinese hamster ovary (CHO) cells. TB cells, CHO cells, erythrocytes, and isolated plasma membranes from CHO cells were labelled with 5-doxylstearic acid and the peak to peak linewidths of the central line of the resulting ESR spectra were measured. The measured line widths were linearly dependent on the amount of 5-doxylstearic acid incorporated into the sample over the range of 0-0.18 mol nitroxide per mol lipid. In erythrocytes, the relationship between linewidths approximated a linear function at lower concentrations of 5-doxylstearic acid, up to 0.07 mol nitroxide per mol lipid. The amount of broadening of the central line for a given amount of 5-doxylstearic acid was far less for intact cells than for either erythrocytes or plasma membrane, indicating that the 5-doxylstearic acid samples a much larger lipid pool in the intact cells. With the broad assumption that the mobility of the 5-doxylstearic acid is similar in different membranes, the size of the lipid pool sampled by 5-doxylstearic acid is approximately equal to the total cellular lipid in intact cells. If a given concentration of 5-doxylstearic acid sampled only the plasma membrane of TB or CHO cells, we would expect to see a linewidth corresponding to a 12-20-fold greater local concentration of 5-doxylstearic acid than was observed, since the plasma membranes of CHO and TB cells represent only 5-8 percent of the total cellular lipid. Therefore, the 5-doxylstearic acid must distribute into most or all cellular membranes of intact cells and is not localized in the plasma membrane alone.  相似文献   

13.
F Schroeder  G Nemecz 《Biochemistry》1989,28(14):5992-6000
The fluorescent sterol dehydroergosterol was used as a cholesterol analogue in conjunction with multifrequency phase and modulation (1-250 MHz) fluorometry to examine whether sterols (1) interact preferentially with fluid- or solid-phase phospholipids and (2) interact preferentially with sphingomyelin in phase-separated or phase-miscible cosonicated phospholipid membranes. Cosonicated small unilamellar vesicles (SUV) were produced by mixing lipids in organic solvents, drying the mixture, adding buffer, sonicating, and separating SUV. Phospholipids of synthetic as well as biological origin were utilized. In phase-separated, cosonicated SUV of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC, 1:1 molar ratio), the fluorescent sterol (0.5 mol %) interacted preferentially with the fluid-phase lipid (partition coefficient, Kf/s = 2.6-3.4) according to four criteria. First, dehydroergosterol detected only the phase transition of DMPC, the phospholipid with the lower phase transition temperature. Second, the dehydroergosterol fluorescence polarization, limiting anisotropy, order parameter, and rotational relaxation time in the cosonicated vesicle were similar to those of dehydroergosterol in SUV composed only of DMPC. Third, the number of dehydroergosterol fluorescence lifetime components as well as the distribution in the cosonicated SUV was similar to that of dehydroergosterol in SUV composed of DMPC. Fourth, dehydroergosterol concentration-dependent self-quenching was detected in DSPC SUV at much lower dehydroergosterol concentration than in DMPC SUV. Preference of dehydroergosterol for fluid-phase lipids was also observed by monitoring dehydroergosterol exchange between individually sonicated DMPC SUV and DSPC SUV after the two types of vesicles were mixed in equal proportions. In these SUV mixtures, the dehydroergosterol also partitioned into the more fluid SUV, 99:1.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
Y Barenholz  N F Moore  R R Wagner 《Biochemistry》1976,15(16):3563-3570
The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.  相似文献   

15.
Small unilamellar vesicles (SUV) were prepared from the total lipid extract of Mycoplasma capricolum. The SUV were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C, and in the presence of 5% polyethylene glycol, an increase in the R18 fluorescence with time was observed when the R18-labeled SUV were introduced to a native M. capricolum cell suspension. The fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes of M. capricolum, was interpreted as a result of lipid mixing during fusion between the SUV and the mycoplasma cells. The presence of cholesterol in the SUV was found to be obligatory to allow SUV-mycoplasma fusion to occur. Adaptation of M. capricolum cells to grow in a medium containing low cholesterol concentration provided cells in which the unesterified cholesterol content was as low as 17 micrograms/mg cell protein. The fusion activity of the adapted cells was very low or nonexistent. Nonetheless, when an early exponential phase culture of the adapted cells was transferred to a cholesterol-rich medium, the cells accumulated cholesterol and regained their fusogenic activity. The cholesterol requirement for fusion in the target mycoplasma membrane was met by a variety of planar sterols having a free beta-hydroxyl group, but differing in the aliphatic side chain, e.g., beta-sitosterol or ergosterol, even though these sterols, having a bulky side chain, are preferentially localized in the outer leaflet of the lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

16.
Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the 'bound' state and the 'free' state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.  相似文献   

17.
J R Wiener  R Pal  Y Barenholz  R R Wagner 《Biochemistry》1985,24(26):7651-7658
In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

18.
The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes®), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum corneum, corroborating the first assumption. To confirm the second postulate, we compared vesicle size in the starting suspension and in the blood after non-invasive transcutaneous aggregate delivery. Size exclusion chromatograms of sera from the mice that received ultradeformable vesicles on the skin were undistinguishable from the results measured with the original vesicle suspension. Taken together, the results support our previous postulate that ultradeformable vesicles penetrate the skin intact, that is, without permanent disintegration.  相似文献   

19.
Effective charge of melittin upon interaction with POPC vesicles   总被引:1,自引:0,他引:1  
The binding of bee venom melittin to small unilamellar vesicles and large nonsonicated multilamellar bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was studied by means of circular dichroism, 31P-NMR and electrophoretic mobility. The melittin binding isotherm for small unilamellar vesicles (SUV) could be described by a partition equilibrium with Kp = (6 +/- 1).10(4) M-1. Electrostatic effects were taken into account by means of the Gouy-Chapman theory. Combining the partition equilibrium with the Gouy-Chapman analysis suggested an effective charge for melittin of Zp = 1.9, which is lower than the true electric charge of 5-6. The variation of the 31P-NMR signal of SUV showed the change in potential at the phosphodiester moiety of the lipid upon addition of melittin. This potential change was lower than that for an ion with an electrical charge of 5-6 and corresponded to a charge of 1.5. Electrophoretic mobility measurements with multilamellar vesicles confirmed the charge reduction effect. These experimental results show that the use of the simple Gouy-Chapman theory requires an effective electrical charge of the melittin which is lower than the formal charge.  相似文献   

20.
Effects of apolipoproteins on the kinetics of cholesterol exchange   总被引:1,自引:0,他引:1  
The effects of apolipoproteins on the kinetics of cholesterol exchange have been investigated by monitoring the transfer of [14C]cholesterol from donor phospholipid/cholesterol complexes containing human apolipoproteins A, B, or C. Negatively charged discoidal and vesicular particles containing purified apolipoproteins complexed with lipid (75 mol % egg PC, 15 mol % dicetyl phosphate, and 10 mol % cholesterol) and a trace of [14C]cholesterol were incubated with a 10-fold excess of neural, acceptor, small unilamellar vesicles (SUV; 90 mol % egg PC and 10 mol % cholesterol). The donor and acceptor particles were separated by chromatography on DEAE-Sepharose, and the rate of movement of labeled cholesterol was analyzed as a first-order exchange process. The kinetics of exchange of cholesterol from both vesicular and discoidal complexes that contain apoproteins are consistent with an aqueous diffusion mechanism, as has been established previously for PC/cholesterol SUV. The addition of 2-3 molecules of apo A-I to a donor SUV does not significantly alter the half-time (t1/2), which is 80 +/- 9 min at 37 degrees C. However, addition of 5-12 apo A-I molecules progressively decreases t1/2 from 65 +/- 2 to 45 +/- 4 min. This enhancement in the rate of desorption of cholesterol molecules is presumed to arise from the creation of packing defects at boundaries around the apoprotein molecules, which are intercalated among the phospholipid and cholesterol molecules in the surface of the donor SUV.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

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