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Influence of α-Tocopherol Incorporation on Ca2+-Induced Fusion of Phosphatidylserine Vesicles
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 333, No. 2, September 15, pp. 394–400, 1996 Article No. 0406
Influence of a-Tocopherol Incorporation on Ca2/-Induced Fusion of Phosphatidylserine Vesicles Francisco J. Aranda,1 M. Paz Sa´nchez-Migallo´n, and Juan C. Go´mez-Ferna´ndez Departamento de Bioquı´mica y Biologı´a Molecular ‘‘A,’’ Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30080, Murcia, Spain
Received February 15, 1996, and in revised form April 25, 1996
The effect of a-tocopherol on the Ca2/-induced fusion of large unilamellar phosphatidylserine vesicles has been investigated. Mixing of aqueous vesicle contents was followed continually with the terbium–dipicolinic acid (Tb–DPA) assay, while the dissociation of preencapsulated Tb–DPA complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure phosphatidylserine and phosphatidylserine containing 2, 5, and 10 mol % of a-tocopherol were employed at different Ca2/ concentrations. The presence of low amounts of a-tocopherol decreased the initial rate of fusion without changing the Ca2/ threshold concentration. The reduction of the initial rate of fusion was proportional to the amount of a-tocopherol present in the bilayer. An a-tocopherol concentrationdependent decrease of both the initial rate and the final extent of release of vesicle contents was also observed. This effect was more evident as more a-tocopherol was incorporated in the bilayer, so that in the presence of 10 mol % of a-tocopherol no significant release was observed after 5 min. The stabilization of the vesicular structure exerted by a-tocopherol was responsible for the apparent increase of the fluorescence intensity of the Tb–DPA complex at later stages of the process. The results reflect a perturbation of the membrane by low concentrations of a-tocopherol which may account for a number of biological effects of this vitamin, not related to its antioxidant role. q 1996 Academic Press, Inc.
Key Words: a-tocopherol; phosphatidylserine; fusion.
a-Tocopherol is the most active and abundant compound with vitamin E biological activity (1). a-Tocopherol has been used widely in the therapy of various clinical conditions and the prevention of degenerative 1
To whom correspondence should be addressed.
diseases (2). It is generally accepted that a very important function of a-tocopherol in membranes is to protect unsaturated lipids from oxidation (3, 4). In addition to its role as a natural antioxidant, a-tocopherol has been shown to affect membrane structure and dynamics, suggesting a role for the vitamin in the stabilization of membranes (5–9). In order to understand the function of a-tocopherol at the molecular level, it is important to study its interaction with membrane components, and specifically with lipids. In this sense, a wide range of physical techniques have been used to ascertain the location of atocopherol in the membrane and its interaction with lipids. These studies included the application of differential scanning calorimetry, electron spin resonance, fluorescence spectroscopy, nuclear magnetic resonance, and Fourier transform infrared spectroscopy (10–23). From these studies it has been concluded that a-tocopherol is located within the membrane with the polar chromanol group near the surface and the hydrophobic tail extending toward the center of the bilayer. Increasing the concentration of a-tocopherol has been shown to progessively broaden and lower the onset temperature of the gel to liquid crystalline phase transition of saturated phospholipids. a-Tocopherol partitioned into the most fluid domain when incorporated in membranes with different composition, increasing ordering and decreasing fluidity of membranes in the crystalline state. Recently a role for a-tocopherol in the modulation of lipid polymorphism has been proposed on the basis of its ability to promote hexagonal HII structures in phosphatidylethanolamine bilayers (24) and to stabilize the bilayer phase when interacting with natural micelle-forming single-chain phospholipids (9). Membrane fusion is a fundamental event in many subcellular and cellular functions. It is of crucial importance in processes such as endocytosis, exocytosis, and fertilization (25, 26). Although the role of Ca2/ as an inducer of fusion is well established in certain cases,
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such as the exocytotic release of cathecholamines (27), the mechanism of these fusion events and their control still remains to be fully elucidated. The transient character of fusion and the chemical and structural complexity of biological membranes have seriously complicated mechanistic studies. Therefore, studies on membrane fusion have resorted to simple model membrane systems, such as divalent cation-induced fusion of phospholipid vesicles containing acidic phospholipids (28). While the Ca2/-induced fusion of PS vesicles has been studied extensively and the interaction of Ca2/ with PS vesicles is well understood, information about the involvement of additional factors in the modulation of Ca2/-induced fusion in biological membranes is limited. Since a-tocopherol is known to be an indispensable lipid component of biological membranes with important biological activities, we decided to investigate the influence of the presence of a-tocopherol on phospholipid vesicle fusion. External addition of a-tocopherol has been reported to fuse erythrocytes (29); however, a-tocopherol has been shown to be ineffective in fusing chromaffin granules aggregated by synexin (30) and to inhibit platelet aggregation (31, 32). More recently it has been reported that a-tocopherol produced a decrease in the aggregation rate of phosphatidylcholine vesicles containing free oleic acid (33). To get insight into the influence of a-tocopherol on vesicle fusion, we have investigated the effect of incorporation of a-tocopherol on Ca2/-induced fusion of phosphatidylserine (PS)2 vesicles. The terbium–dipicolinic acid (Tb–DPA) assay (34), which monitors the mixing of aqueous contents and can also be used to measure release of vesicle contents (35), was used to follow the process of fusion. We present experiments which indicate that the presence of a-tocopherol inhibits drastically the Ca2/-induced fusion of PS vesicles. The results are discussed in the light of the possible role of a-tocopherol in membrane fusion, and its relation to other membrane effects which do not involve an antioxidant mechanism of action.
on the assay to be performed (mixing or release of aqueous contents as specified below) by vortex mixing. The multilamellar vesicle suspension was frozen in liquid nitrogen and thawed in a 307C water bath five times. Large unilamellar vesicles were obtained by extrusion through two stacked polycarbonate filters (Nuclepore, 0.1-mm pore size) using an Extruder (Lipex Biomembranes Inc., Vancouver, Canada) essentially as described by Hope et al. (36). Lipid vesicle samples were analyzed for organic phosphorous according to the method of Bo¨tcher et al. (37). Evaluation of lipid peroxidation showed no significant production of peroxides during the time scale of the fusion assays both in the absence and in the presence of a-tocopherol. Tb–DPA fusion and leakage assay. Mixing of aqueous vesicle contents was measured by using the Tb–DPA fusion assay as described by Wilschut et al. (34). Vesicles were made in (a) 2.5 mM TbCl3 and 50 mM sodium citrate, (b) 50 mM DPA (sodium salt) and 20 mM NaCl, or (c) 2.5 mM TbCl3 , 25 mM sodium citrate, 25 mM DPA, and 10 mM NaCl. All solutions were buffered with 10 mM Hepes (pH 7.4). Unencapsulated material was removed by gel filtration on Sephadex G-50 using 1 mM EDTA, 100 mM NaCl, and 10 mM Hepes (pH 7.4) as elution buffer. For purposes of calibration, portions of the Tb-vesicles prepared in buffer (a) were rechromatographed with an EDTA-free buffer in order to remove the external EDTA. Fusion of the vesicles was measured by monitoring the formation of the fluorescent Tb–(DPA)30 3 complex upon mixing of the aqueous contents of Tb- and DPA-containing vesicles. Fusion measurements were carried out in a cuvette containing 2 ml of a 1:1 mixture of Tbvesicles (type a) and DPA-vesicles (type b) at a final concentration of 50 mM phospholipid in 0.1 mM EDTA, 100 mM NaCl, and 10 mM Hepes (pH 7.4). Fluorescence was monitored in a Shimadzu RF-540 spectrofluorometer. The temperature was maintained at 257C, and the solution in the cuvette was continuously mixed by stirring. Appropriate amounts of CaCl2 were injected via Hamilton syringes directly into the cuvette. The Tb–DPA complex was excited at 276 nm and emission was measured at 545 nm with a cutoff filter (selecting wavelengths higher than 520 nm). a-Tocopherol did not interfere with the fluorescence properties of the Tb–DPA complex. The amount of passive leakage in all samples was always less than 3% of the amount of Tb initially present in the vesicles. This latter value was obtained by addition of sodium cholate (1%, w/v) to the cuvette, causing all the Tb to complex with DPA. Subtraction of the amount of passive leakage from the amount of Tb initially present in the vesicles yielded the amount of Tb present in the vesicles at the time of cholate addition, the ‘‘100% Tb fluorescence value.’’ The release of internal aqueous contents during fusion was measured by monitoring the dissociation of preencapsulated Tb–DPA complex (type c) vesicles as described by Bentz et al. (35), under the same conditions as in the mixing of aqueous contents experiments. Vesicle aggregation was followed by changes in 907 light scattering at 400 nm using a BioLogic Modular Optical System (Grenoble, France).
MATERIALS AND METHODS
RESULTS
Chemicals. Spinal cord PS was obtained from Lipid Products (Surrey, UK), a-tocopherol and DPA were from Sigma, (Poole, Dorset, UK) and TbCl3r6 H2O was obtained from Janssen Chimica (Belgium). All other reagents were of the highest purity available.
Both aggregation and actual fusion contribute to the overall process of fusion (38). Figure 1 shows that the presence of increasing concentrations of a-tocopherol did not affect the initial rate of vesicle aggregation. Therefore, any observed differences in the rate of fusion are not due to changes in aggregation rate but to changes in the fusion process per se. The fusion of PS vesicles can be monitored conveniently by using the fluorescent assay described above (34). This assay monitors the mixing of aqueous vesicle contents between two populations of phospholipid vesicles containing TbCl3 and DPA. The formation of the
Preparation of large unilamellar vesicles. Chloroform solutions containing phospholipid and the appropriate amount of a-tocopherol when necessary, to give different final concentrations as indicated in each case, were combined and dried under a nitrogen stream and stored for 3 h under vacuum to remove the last traces of solvent. The dry lipid film was dispersed in 1 ml of aqueous buffer depending
2
Abbreviations used: PS, phosphatidylserine; Tb–DPA, terbium– dipicolinic acid.
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FIG. 1. Time-dependent vesicle aggregation for pure phosphatidylserine vesicles (a) and phosphatidylserine containing 2 mol % (b), 5 mol % (c), and 10 mol % (d) of a-tocopherol. Calcium was added at Time 0 at 8 mM final concentration. 100% light scattering corresponds to the maximum extent of aggregation obtained with pure phosphatidylserine vesicles.
Tb–DPA complex produces an enhancement in Tb fluorescence. Formation of the fluorescent Tb–DPA complex in the external medium is prevented by the presence of EDTA and Ca2/, which rapidly dissociates the complex; consequently, only the mixing of vesicular contents, separated from the outside medium, is registered as fusion. Leakage of the complex from the vesicles causes a rapid decrease in the fluorescence signal. The actual extent of vesicle content leakage can be determined in parallel vesicle preparations by the decrease of fluorescence intensity that occur upon release and dissociation of Tb–DPA complex entrapped in vesicles. The time course of 8 mM Ca2/-induced fusion, measured as mixing of aqueous contents, of PS and a-tocopherol containing PS vesicles is depicted in Fig. 2. In agreement with a previous report (34), in the absence of Ca2/, the Tb fluorescence of pure PS vesicles remained constant at a very low level. After addition of Ca2/ to the vesicle suspension, the Tb fluorescence intensity increased sharply until a maximum was reached which was followed by a relatively slow decrease (Fig. 2, trace a). The fusion of a-tocopherol containing PS vesicles showed the same behavior; however, clear differences were observed concerning the rate and the extent of the process. The rate of mixing of aqueous contents in the case of PS vesicles which contained different amounts of a-tocopherol (Fig. 2, traces b, c, and d) was slower than that of control vesicles (Fig. 2, trace a). While the apparent maximal fluorescence intensity of the Tb–DPA complex for PS vesicles containing 2 mol % a-tocopherol was lower than that of pure PS, the presence of 5 and 10 mol % a-tocopherol produced a progressive enhancement of the apparent maximal
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fluorescence intensity, which was higher than that of control vesicles. It is known that the initial rate and the apparent maximal fluorescence intensity for PS vesicles are markedly dependent on the Ca2/ concentration in the medium (33). In Fig. 3, the initial rates of the Tb fluorescence increase (Fig. 3A) and the apparent maximal fluorescence intensity (Fig. 3B) for PS and a-tocopherol containing PS vesicles are plotted against the Ca2/ concentration. From Fig. 3A, an apparent Ca2/ threshold concentration can be obtained by extrapolation to zero of the steeply increasing part of the curve, which relates the initial rates of fusion to the Ca2/ concentration. For control PS vesicles an apparent threshold concentration of 2.3 mM Ca2/ was observed in close agreement with that previously reported (34). The initial rate of fusion increased with increasing Ca2/ concentration up to about 6–8 mM, at higher Ca2/ concentrations an apparent saturation was observed. The presence of a-tocopherol did not significantly change the apparent Ca2/ threshold concentration; however the presence of a-tocopherol produced a significant reduction of the initial rate of fusion. This reduction was proportional to the amount of a-tocopherol present in the bilayer, so that increasing the concentration of atocopherol produced a further decrease of the rate of fusion such that the sample containing 10 mol % atocopherol showed, at 8 mM Ca2/, a threefold reduction of the rate of fusion when compared to control vesicles containing no a-tocopherol. Figure 3B shows the aparent maximal fluorescence intensity against the Ca2/ concentration for PS vesicles and those containing increasing amounts of a-tocopherol. Increasing the presence of a-tocopherol in PS vesicles produced an increase in the apparent maximal fluorescence intensity
FIG. 2. Ca2/-induced fusion of vesicle made of pure PS (a) and PS containing 2 mol % (b), 5 mol % (c), and 10 mol % (d) of a-tocopherol, as measured by the Tb–DPA assay. Calcium was added at Time 0 at 8 mM final concentration. Fusion is presented as the percentage of encapsulated Tb complexed to DPA.
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FIG. 3. Dependence of the initial rate of fusion (A) and apparent maximal fluorescence intensity of the Tb–DPA complex (B) measured as mixing of aqueous contents on the Ca2/ concentration for vesicles made of pure PS (s) and PS containing 2 mol % (l), 5 mol % (h), and 10 mol % (j) of a-tocopherol.
at concentrations of Ca2/ higher than 4 mM, such that the sample containing 10 mol % a-tocopherol in the presence of 8 mM Ca2/ showed almost a twofold increase in fluorescence intensity when compared to control vesicles. It is known that the apparent maximal fluorescence intensity and the eventual decrease in the Tb fluorescence observed in the mixing of aqueous contents (Fig. 2) depend on the release of vesicle contents and dissociation of the Tb–DPA complex in the external medium. Although the initial stage of Ca2/-induced PS vesicle fusion is nonleaky (39) the secondary release of contents affects the apparent maximal fluorescence intensity of the complex. Therefore, we next studied the release of vesicle contents in a direct way to complement the measurements of mixing of aqueous contents and also to obtain a picture of the stability of the process. The time course of release of contents from PS vesicles and a-tocopherol containing vesicles at 8 mM Ca2/, is depicted in Fig. 4. From the comparison between Figs. 2 and 4 it is seen that the mixing of aqueous vesicular contents of PS vesicles (Fig. 2, trace a) clearly preceded that of the release of vesicles contents to the external medium (Fig. 4, trace a), in accordance to that previously reported (34, 39), indicating that during the initial stages of the process there was no dectable release at all. It is in the latter stages of the process that release of contents occurred, reaching almost 90% at 5 min (Fig. 4, trace a). An interesting finding is that the presence of low amounts of a-tocopherol (such as 2 mol %) produced a decrease of the rate of release of contents and more important a decrease of the final extent of release. Increasing the amount of a-tocopherol present in PS bilayers produced a further decrease in the release of contents reaching only half of the release observed in control vesicles at longer times. The release
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of contents from these vesicles at 5 min after addition of Ca2/ is plotted against the Ca2/ concentration in Fig. 5. The extent of release of contents increased with increasing Ca2/, reaching nearly 90% in the case of PS vesicles at the highest Ca2/ concentration tested. The presence of a-tocopherol produced a decrease in the extent of release at all Ca2/ concentrations, this effect being more evident as more a-tocopherol is incorporated in the bilayer; it can be seen that PS vesicles released about 60% of their contents at 5 min after addition of 4 mM Ca2/. However, in the presence of 2 mol % a-tocopherol they had a 27% release; with 5 mol % a-tocopherol only a 4% release was observed, and in
FIG. 4. Ca2/-induced release of aqueous contents from vesicles made of pure PS (a) and PS containing 2 mol % (b), 5 mol % (c), and 10 mol % (d) of a-tocopherol. The release was measured with vesicles containing the Tb–DPA complex and shows the percentage of the fluorescent complex that has dissociated due to leakage from, and the entry of medium into the vesicles. Calcium was added at Time 0 at 8 mM final concentration.
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FIG. 5. Dependence of the release of aqueous contents on the Ca2/ concentration for vesicles made of pure PS (s) and PS containing 2 mol % (l), 5 mol % (h), and 10 mol % (j) of a-tocopherol, measured as the percentage of release at 5 min after the addition of Ca2/.
the presence of 10 mol % a-tocopherol no significant release was seen at 5 min. DISCUSSION
In this study, we show that incorporation of a-tocopherol into large unilamellar PS vesicles inhibited the Ca2/-induced fusion of these vesicles. This finding is on-line with the suggestion that a-tocopherol may have a role in the stabilization of membranes against fusion. We have observed the same behavior for the Ca2/induced fusion in pure PS vesicles as in a-tocopherol containing PS vesicles; hence it is likely that the presence of a-tocopherol does not alter the fusion mechanism. The initial rate of Ca2/-induced fusion decreased as the presence of a-tocopherol was increased. However, it was found that the apparent maximal fluorescence intensity of the Tb–DPA complex was higher in the vesicles with higher incorporation of a-tocopherol. It could be that this higher maximal fluorecence intensity obtained in the presence of a-tocopherol reflects a stimulation of the process by the vitamin molecule. This would contradict the inhibition exerted by a-tocopherol on the rate of the process. It is known that the maximal fluorescence intensity is closely related to the release of aqueous contents responsible for the eventual decrease in Tb fluorescence intensity (28). Hence an increase in the apparent maximal fluorescence intensity could actually reflect a slowing of the release instead of a stimulation of the mixing of aqueous contents. In fact, when we studied the Ca2/-induced release of aqueous contents, we found that the presence of a-tocopherol inhibited this release, thus stabilizing the PS vesicles and producing an apparent increase in the maximal fluorescence intensity of the complex. Since the
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leakage of contents marks the first stages of the collapse of the fused vesicles into large nonvesicular structures, the inhibition of this leakage underlines an important effect of a-tocopherol as a stabilizer of the phospholipid vesicular structure. The observed decrease of the initial rate of fusion and release of contents in the presence of a-tocopherol may be related to several factors. The first is the dilution of PS molecules by the inclusion of a-tocopherol. As a result, the possibility of formation of intermembrane contacts leading to fusion may be lowered. Also a-tocopherol may alter the spacing between PS molecules and affect the coordination complex between Ca2/ and PS. In fact, we have recently found that a-tocopherol produced a decrease in the the number of binding sites and a reduction in the apparent affinity for Ca2/ by dimyristoylphosphatidylserine (40). The second factor is the reduced ‘‘fluidity’’ or increase in order of membranes containing a-tocopherol, as measured by fluorescence polarization (41), electron spin resonance (16) and FT-IR spectroscopy (11), which implies a reduced mobility. Membranes in the fluid state are considerably more prone to fusion than those in the solid phase (42). The reported decrease in the fluidity of the membrane by a-tocopherol may contribute to the lower fusion rate and release of contents. In this report a-tocopherol behaves like cholesterol, which has been shown to reduce the ‘‘fluidity’’ of membranes (43) and also to similarly decrease the overall rate of Ba2/- and Ca2/-induced fusion in PS vesicles (44, 45). However, clear differences can be observed between cholesterol and a-tocopherol: first, the observation that cholesterol, as opposed to a-tocopherol, did not reduce the leakage of internal contents during fusion (45), and second, that the effects of cholesterol are observed at considerably higher concentration (ú20 mol %) than those reported here for a-tocopherol. In addition to that, the effect of cholesterol seems to be somehow more complex, since under certain conditions it can also increase the rate of fusion (44, 45). It is interesting to note that a-tocopherol has been reported to preferentially partition into the most fluid domain when incorporated into mixed phospholipid vesicles (12). This was considered to be important because it would cause a-tocopherol to be associated to more unsaturated fatty acyl chains, hence facilitating its peroxidative-protecting task. Considering the results shown here, this location of a-tocopherol would also be important because these more fluid areas are precisely those more prone to undergo fusion and hence would facilitate its possible role as a membrane stabilizer against fusion. A third factor is the possibility of the increased hydration of the membrane surface with increasing atocopherol. Studies on PC/a-tocopherol vesicles by FTIR spectroscopy indicated that the presence of a-tocopherol produced a shift of the C|O stretching mode
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to lower wavenumbers (11); this suggests an increase in the proportion of carbonyl groups of the lipid which are in a more hydrated form. This hypothetically higher hydration would make more difficult a complete dehydration by Ca2/ and the formation of packing defects. This effect of a-tocopherol on PS bilayers which leads to a stabilization of the membrane against fusion may have the same origin as other effects described for the vitamin on membranes. In this respect, a-tocopherol has been shown to inhibit protein kinase C activity (46) which is known to require PS for activation. Inversely, 1,2-dioleoylglycerol, which is a well known activator of protein kinase C (47), has been shown to stimulate Ca2/-induced PS vesicle fusion (48, 49). It might be suggested that the perturbation exerted by a-tocopherol on PS molecules, which is responsible for the stabilization of membrane against fusion, could also affect other roles of PS in membrane function. We have described, in this context, that a-tocopherol decreases the affinity of PS for 45Ca2/ (40), stressing again that this vitamin molecule perturbs the lipid–water interface of the membrane, reducing the capacity of PS to interact with Ca2/ and participate in membrane fusion. In summary, a-tocopherol has been shown to produce a decrease in the rate of fusion and to inhibit the release of contents of Ca2/-induced PS vesicle fusion. This work reinforces the notion that a-tocopherol has the important capacity of acting as membrane stabilizer in addition to its well known function as a natural antioxidant.
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ACKNOWLEDGMENTS This work was supported by Grants PB 95/08 from Comisio´n Interministerial de Ciencia y Tecnologı´a, Spain, and PCT 93/22 from Direccio´n General de Educacio´n y Universidad, Comunidad Auto´noma de Murcia, Spain. F.J.A. thanks Dr. A. Ortiz for valuable discussion on lipid peroxidation.
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Bentz, J., and Du¨zgu¨nez, N. (1988) Biochim. Biophys. Acta 946, 405–416. Bental, M., Wilschut, J., Scholma, J., and Nir, S. (1987) Biochim. Biophys. Acta 898, 239–247. ¨ zer, N. K., Palozza, P., Boscoboinik, D., and Azzi, A. (1993) O FEBS Lett. 322, 307–310. Zidovetzki, R., and Lester, D. S. (1992) Biochim. Biophys. Acta 1134, 261–272. Ortiz, A., Aranda, F. J., Villalaı´n, J., San Martı´n, C., Micol, V., and Go´mez-Ferna´ndez, J. C. (1992) Chem. Phys. Lipids 62, 215– 224. Sa´nchez-Migallo´n, M. P., Aranda, F. J., and Go´mez-Ferna´ndez, J. C. (1995) Biophys. J. 68, 558–566.
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41. Urano, S., Inomori, Y., Sugawara, T., Kato, Y., Kitachara, M., Hasegawa, Y., Matsuo, M., and Mukai, K. (1992) J. Biol. Chem. 267, 18365–18370.
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42. Wilschut, J., Du¨zgu¨nez, N., Hoekstra, D., and Papahadjopoulos, D. (1985) Biochemistry 24, 8–14. 43. Guyer, W., and Bloch, K. (1983) Chem. Phys. Lipids 33, 313– 322.
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Vol. 333, No. 2, September 15, pp. 394–400, 1996 Article No. 0406
Influence of a-Tocopherol Incorporation on Ca2/-Induced Fusion of Phosphatidylserine Vesicles Francisco J. Aranda,1 M. Paz Sa´nchez-Migallo´n, and Juan C. Go´mez-Ferna´ndez Departamento de Bioquı´mica y Biologı´a Molecular ‘‘A,’’ Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30080, Murcia, Spain
Received February 15, 1996, and in revised form April 25, 1996
The effect of a-tocopherol on the Ca2/-induced fusion of large unilamellar phosphatidylserine vesicles has been investigated. Mixing of aqueous vesicle contents was followed continually with the terbium–dipicolinic acid (Tb–DPA) assay, while the dissociation of preencapsulated Tb–DPA complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure phosphatidylserine and phosphatidylserine containing 2, 5, and 10 mol % of a-tocopherol were employed at different Ca2/ concentrations. The presence of low amounts of a-tocopherol decreased the initial rate of fusion without changing the Ca2/ threshold concentration. The reduction of the initial rate of fusion was proportional to the amount of a-tocopherol present in the bilayer. An a-tocopherol concentrationdependent decrease of both the initial rate and the final extent of release of vesicle contents was also observed. This effect was more evident as more a-tocopherol was incorporated in the bilayer, so that in the presence of 10 mol % of a-tocopherol no significant release was observed after 5 min. The stabilization of the vesicular structure exerted by a-tocopherol was responsible for the apparent increase of the fluorescence intensity of the Tb–DPA complex at later stages of the process. The results reflect a perturbation of the membrane by low concentrations of a-tocopherol which may account for a number of biological effects of this vitamin, not related to its antioxidant role. q 1996 Academic Press, Inc.
Key Words: a-tocopherol; phosphatidylserine; fusion.
a-Tocopherol is the most active and abundant compound with vitamin E biological activity (1). a-Tocopherol has been used widely in the therapy of various clinical conditions and the prevention of degenerative 1
To whom correspondence should be addressed.
diseases (2). It is generally accepted that a very important function of a-tocopherol in membranes is to protect unsaturated lipids from oxidation (3, 4). In addition to its role as a natural antioxidant, a-tocopherol has been shown to affect membrane structure and dynamics, suggesting a role for the vitamin in the stabilization of membranes (5–9). In order to understand the function of a-tocopherol at the molecular level, it is important to study its interaction with membrane components, and specifically with lipids. In this sense, a wide range of physical techniques have been used to ascertain the location of atocopherol in the membrane and its interaction with lipids. These studies included the application of differential scanning calorimetry, electron spin resonance, fluorescence spectroscopy, nuclear magnetic resonance, and Fourier transform infrared spectroscopy (10–23). From these studies it has been concluded that a-tocopherol is located within the membrane with the polar chromanol group near the surface and the hydrophobic tail extending toward the center of the bilayer. Increasing the concentration of a-tocopherol has been shown to progessively broaden and lower the onset temperature of the gel to liquid crystalline phase transition of saturated phospholipids. a-Tocopherol partitioned into the most fluid domain when incorporated in membranes with different composition, increasing ordering and decreasing fluidity of membranes in the crystalline state. Recently a role for a-tocopherol in the modulation of lipid polymorphism has been proposed on the basis of its ability to promote hexagonal HII structures in phosphatidylethanolamine bilayers (24) and to stabilize the bilayer phase when interacting with natural micelle-forming single-chain phospholipids (9). Membrane fusion is a fundamental event in many subcellular and cellular functions. It is of crucial importance in processes such as endocytosis, exocytosis, and fertilization (25, 26). Although the role of Ca2/ as an inducer of fusion is well established in certain cases,
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such as the exocytotic release of cathecholamines (27), the mechanism of these fusion events and their control still remains to be fully elucidated. The transient character of fusion and the chemical and structural complexity of biological membranes have seriously complicated mechanistic studies. Therefore, studies on membrane fusion have resorted to simple model membrane systems, such as divalent cation-induced fusion of phospholipid vesicles containing acidic phospholipids (28). While the Ca2/-induced fusion of PS vesicles has been studied extensively and the interaction of Ca2/ with PS vesicles is well understood, information about the involvement of additional factors in the modulation of Ca2/-induced fusion in biological membranes is limited. Since a-tocopherol is known to be an indispensable lipid component of biological membranes with important biological activities, we decided to investigate the influence of the presence of a-tocopherol on phospholipid vesicle fusion. External addition of a-tocopherol has been reported to fuse erythrocytes (29); however, a-tocopherol has been shown to be ineffective in fusing chromaffin granules aggregated by synexin (30) and to inhibit platelet aggregation (31, 32). More recently it has been reported that a-tocopherol produced a decrease in the aggregation rate of phosphatidylcholine vesicles containing free oleic acid (33). To get insight into the influence of a-tocopherol on vesicle fusion, we have investigated the effect of incorporation of a-tocopherol on Ca2/-induced fusion of phosphatidylserine (PS)2 vesicles. The terbium–dipicolinic acid (Tb–DPA) assay (34), which monitors the mixing of aqueous contents and can also be used to measure release of vesicle contents (35), was used to follow the process of fusion. We present experiments which indicate that the presence of a-tocopherol inhibits drastically the Ca2/-induced fusion of PS vesicles. The results are discussed in the light of the possible role of a-tocopherol in membrane fusion, and its relation to other membrane effects which do not involve an antioxidant mechanism of action.
on the assay to be performed (mixing or release of aqueous contents as specified below) by vortex mixing. The multilamellar vesicle suspension was frozen in liquid nitrogen and thawed in a 307C water bath five times. Large unilamellar vesicles were obtained by extrusion through two stacked polycarbonate filters (Nuclepore, 0.1-mm pore size) using an Extruder (Lipex Biomembranes Inc., Vancouver, Canada) essentially as described by Hope et al. (36). Lipid vesicle samples were analyzed for organic phosphorous according to the method of Bo¨tcher et al. (37). Evaluation of lipid peroxidation showed no significant production of peroxides during the time scale of the fusion assays both in the absence and in the presence of a-tocopherol. Tb–DPA fusion and leakage assay. Mixing of aqueous vesicle contents was measured by using the Tb–DPA fusion assay as described by Wilschut et al. (34). Vesicles were made in (a) 2.5 mM TbCl3 and 50 mM sodium citrate, (b) 50 mM DPA (sodium salt) and 20 mM NaCl, or (c) 2.5 mM TbCl3 , 25 mM sodium citrate, 25 mM DPA, and 10 mM NaCl. All solutions were buffered with 10 mM Hepes (pH 7.4). Unencapsulated material was removed by gel filtration on Sephadex G-50 using 1 mM EDTA, 100 mM NaCl, and 10 mM Hepes (pH 7.4) as elution buffer. For purposes of calibration, portions of the Tb-vesicles prepared in buffer (a) were rechromatographed with an EDTA-free buffer in order to remove the external EDTA. Fusion of the vesicles was measured by monitoring the formation of the fluorescent Tb–(DPA)30 3 complex upon mixing of the aqueous contents of Tb- and DPA-containing vesicles. Fusion measurements were carried out in a cuvette containing 2 ml of a 1:1 mixture of Tbvesicles (type a) and DPA-vesicles (type b) at a final concentration of 50 mM phospholipid in 0.1 mM EDTA, 100 mM NaCl, and 10 mM Hepes (pH 7.4). Fluorescence was monitored in a Shimadzu RF-540 spectrofluorometer. The temperature was maintained at 257C, and the solution in the cuvette was continuously mixed by stirring. Appropriate amounts of CaCl2 were injected via Hamilton syringes directly into the cuvette. The Tb–DPA complex was excited at 276 nm and emission was measured at 545 nm with a cutoff filter (selecting wavelengths higher than 520 nm). a-Tocopherol did not interfere with the fluorescence properties of the Tb–DPA complex. The amount of passive leakage in all samples was always less than 3% of the amount of Tb initially present in the vesicles. This latter value was obtained by addition of sodium cholate (1%, w/v) to the cuvette, causing all the Tb to complex with DPA. Subtraction of the amount of passive leakage from the amount of Tb initially present in the vesicles yielded the amount of Tb present in the vesicles at the time of cholate addition, the ‘‘100% Tb fluorescence value.’’ The release of internal aqueous contents during fusion was measured by monitoring the dissociation of preencapsulated Tb–DPA complex (type c) vesicles as described by Bentz et al. (35), under the same conditions as in the mixing of aqueous contents experiments. Vesicle aggregation was followed by changes in 907 light scattering at 400 nm using a BioLogic Modular Optical System (Grenoble, France).
MATERIALS AND METHODS
RESULTS
Chemicals. Spinal cord PS was obtained from Lipid Products (Surrey, UK), a-tocopherol and DPA were from Sigma, (Poole, Dorset, UK) and TbCl3r6 H2O was obtained from Janssen Chimica (Belgium). All other reagents were of the highest purity available.
Both aggregation and actual fusion contribute to the overall process of fusion (38). Figure 1 shows that the presence of increasing concentrations of a-tocopherol did not affect the initial rate of vesicle aggregation. Therefore, any observed differences in the rate of fusion are not due to changes in aggregation rate but to changes in the fusion process per se. The fusion of PS vesicles can be monitored conveniently by using the fluorescent assay described above (34). This assay monitors the mixing of aqueous vesicle contents between two populations of phospholipid vesicles containing TbCl3 and DPA. The formation of the
Preparation of large unilamellar vesicles. Chloroform solutions containing phospholipid and the appropriate amount of a-tocopherol when necessary, to give different final concentrations as indicated in each case, were combined and dried under a nitrogen stream and stored for 3 h under vacuum to remove the last traces of solvent. The dry lipid film was dispersed in 1 ml of aqueous buffer depending
2
Abbreviations used: PS, phosphatidylserine; Tb–DPA, terbium– dipicolinic acid.
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FIG. 1. Time-dependent vesicle aggregation for pure phosphatidylserine vesicles (a) and phosphatidylserine containing 2 mol % (b), 5 mol % (c), and 10 mol % (d) of a-tocopherol. Calcium was added at Time 0 at 8 mM final concentration. 100% light scattering corresponds to the maximum extent of aggregation obtained with pure phosphatidylserine vesicles.
Tb–DPA complex produces an enhancement in Tb fluorescence. Formation of the fluorescent Tb–DPA complex in the external medium is prevented by the presence of EDTA and Ca2/, which rapidly dissociates the complex; consequently, only the mixing of vesicular contents, separated from the outside medium, is registered as fusion. Leakage of the complex from the vesicles causes a rapid decrease in the fluorescence signal. The actual extent of vesicle content leakage can be determined in parallel vesicle preparations by the decrease of fluorescence intensity that occur upon release and dissociation of Tb–DPA complex entrapped in vesicles. The time course of 8 mM Ca2/-induced fusion, measured as mixing of aqueous contents, of PS and a-tocopherol containing PS vesicles is depicted in Fig. 2. In agreement with a previous report (34), in the absence of Ca2/, the Tb fluorescence of pure PS vesicles remained constant at a very low level. After addition of Ca2/ to the vesicle suspension, the Tb fluorescence intensity increased sharply until a maximum was reached which was followed by a relatively slow decrease (Fig. 2, trace a). The fusion of a-tocopherol containing PS vesicles showed the same behavior; however, clear differences were observed concerning the rate and the extent of the process. The rate of mixing of aqueous contents in the case of PS vesicles which contained different amounts of a-tocopherol (Fig. 2, traces b, c, and d) was slower than that of control vesicles (Fig. 2, trace a). While the apparent maximal fluorescence intensity of the Tb–DPA complex for PS vesicles containing 2 mol % a-tocopherol was lower than that of pure PS, the presence of 5 and 10 mol % a-tocopherol produced a progressive enhancement of the apparent maximal
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fluorescence intensity, which was higher than that of control vesicles. It is known that the initial rate and the apparent maximal fluorescence intensity for PS vesicles are markedly dependent on the Ca2/ concentration in the medium (33). In Fig. 3, the initial rates of the Tb fluorescence increase (Fig. 3A) and the apparent maximal fluorescence intensity (Fig. 3B) for PS and a-tocopherol containing PS vesicles are plotted against the Ca2/ concentration. From Fig. 3A, an apparent Ca2/ threshold concentration can be obtained by extrapolation to zero of the steeply increasing part of the curve, which relates the initial rates of fusion to the Ca2/ concentration. For control PS vesicles an apparent threshold concentration of 2.3 mM Ca2/ was observed in close agreement with that previously reported (34). The initial rate of fusion increased with increasing Ca2/ concentration up to about 6–8 mM, at higher Ca2/ concentrations an apparent saturation was observed. The presence of a-tocopherol did not significantly change the apparent Ca2/ threshold concentration; however the presence of a-tocopherol produced a significant reduction of the initial rate of fusion. This reduction was proportional to the amount of a-tocopherol present in the bilayer, so that increasing the concentration of atocopherol produced a further decrease of the rate of fusion such that the sample containing 10 mol % atocopherol showed, at 8 mM Ca2/, a threefold reduction of the rate of fusion when compared to control vesicles containing no a-tocopherol. Figure 3B shows the aparent maximal fluorescence intensity against the Ca2/ concentration for PS vesicles and those containing increasing amounts of a-tocopherol. Increasing the presence of a-tocopherol in PS vesicles produced an increase in the apparent maximal fluorescence intensity
FIG. 2. Ca2/-induced fusion of vesicle made of pure PS (a) and PS containing 2 mol % (b), 5 mol % (c), and 10 mol % (d) of a-tocopherol, as measured by the Tb–DPA assay. Calcium was added at Time 0 at 8 mM final concentration. Fusion is presented as the percentage of encapsulated Tb complexed to DPA.
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FIG. 3. Dependence of the initial rate of fusion (A) and apparent maximal fluorescence intensity of the Tb–DPA complex (B) measured as mixing of aqueous contents on the Ca2/ concentration for vesicles made of pure PS (s) and PS containing 2 mol % (l), 5 mol % (h), and 10 mol % (j) of a-tocopherol.
at concentrations of Ca2/ higher than 4 mM, such that the sample containing 10 mol % a-tocopherol in the presence of 8 mM Ca2/ showed almost a twofold increase in fluorescence intensity when compared to control vesicles. It is known that the apparent maximal fluorescence intensity and the eventual decrease in the Tb fluorescence observed in the mixing of aqueous contents (Fig. 2) depend on the release of vesicle contents and dissociation of the Tb–DPA complex in the external medium. Although the initial stage of Ca2/-induced PS vesicle fusion is nonleaky (39) the secondary release of contents affects the apparent maximal fluorescence intensity of the complex. Therefore, we next studied the release of vesicle contents in a direct way to complement the measurements of mixing of aqueous contents and also to obtain a picture of the stability of the process. The time course of release of contents from PS vesicles and a-tocopherol containing vesicles at 8 mM Ca2/, is depicted in Fig. 4. From the comparison between Figs. 2 and 4 it is seen that the mixing of aqueous vesicular contents of PS vesicles (Fig. 2, trace a) clearly preceded that of the release of vesicles contents to the external medium (Fig. 4, trace a), in accordance to that previously reported (34, 39), indicating that during the initial stages of the process there was no dectable release at all. It is in the latter stages of the process that release of contents occurred, reaching almost 90% at 5 min (Fig. 4, trace a). An interesting finding is that the presence of low amounts of a-tocopherol (such as 2 mol %) produced a decrease of the rate of release of contents and more important a decrease of the final extent of release. Increasing the amount of a-tocopherol present in PS bilayers produced a further decrease in the release of contents reaching only half of the release observed in control vesicles at longer times. The release
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of contents from these vesicles at 5 min after addition of Ca2/ is plotted against the Ca2/ concentration in Fig. 5. The extent of release of contents increased with increasing Ca2/, reaching nearly 90% in the case of PS vesicles at the highest Ca2/ concentration tested. The presence of a-tocopherol produced a decrease in the extent of release at all Ca2/ concentrations, this effect being more evident as more a-tocopherol is incorporated in the bilayer; it can be seen that PS vesicles released about 60% of their contents at 5 min after addition of 4 mM Ca2/. However, in the presence of 2 mol % a-tocopherol they had a 27% release; with 5 mol % a-tocopherol only a 4% release was observed, and in
FIG. 4. Ca2/-induced release of aqueous contents from vesicles made of pure PS (a) and PS containing 2 mol % (b), 5 mol % (c), and 10 mol % (d) of a-tocopherol. The release was measured with vesicles containing the Tb–DPA complex and shows the percentage of the fluorescent complex that has dissociated due to leakage from, and the entry of medium into the vesicles. Calcium was added at Time 0 at 8 mM final concentration.
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FIG. 5. Dependence of the release of aqueous contents on the Ca2/ concentration for vesicles made of pure PS (s) and PS containing 2 mol % (l), 5 mol % (h), and 10 mol % (j) of a-tocopherol, measured as the percentage of release at 5 min after the addition of Ca2/.
the presence of 10 mol % a-tocopherol no significant release was seen at 5 min. DISCUSSION
In this study, we show that incorporation of a-tocopherol into large unilamellar PS vesicles inhibited the Ca2/-induced fusion of these vesicles. This finding is on-line with the suggestion that a-tocopherol may have a role in the stabilization of membranes against fusion. We have observed the same behavior for the Ca2/induced fusion in pure PS vesicles as in a-tocopherol containing PS vesicles; hence it is likely that the presence of a-tocopherol does not alter the fusion mechanism. The initial rate of Ca2/-induced fusion decreased as the presence of a-tocopherol was increased. However, it was found that the apparent maximal fluorescence intensity of the Tb–DPA complex was higher in the vesicles with higher incorporation of a-tocopherol. It could be that this higher maximal fluorecence intensity obtained in the presence of a-tocopherol reflects a stimulation of the process by the vitamin molecule. This would contradict the inhibition exerted by a-tocopherol on the rate of the process. It is known that the maximal fluorescence intensity is closely related to the release of aqueous contents responsible for the eventual decrease in Tb fluorescence intensity (28). Hence an increase in the apparent maximal fluorescence intensity could actually reflect a slowing of the release instead of a stimulation of the mixing of aqueous contents. In fact, when we studied the Ca2/-induced release of aqueous contents, we found that the presence of a-tocopherol inhibited this release, thus stabilizing the PS vesicles and producing an apparent increase in the maximal fluorescence intensity of the complex. Since the
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leakage of contents marks the first stages of the collapse of the fused vesicles into large nonvesicular structures, the inhibition of this leakage underlines an important effect of a-tocopherol as a stabilizer of the phospholipid vesicular structure. The observed decrease of the initial rate of fusion and release of contents in the presence of a-tocopherol may be related to several factors. The first is the dilution of PS molecules by the inclusion of a-tocopherol. As a result, the possibility of formation of intermembrane contacts leading to fusion may be lowered. Also a-tocopherol may alter the spacing between PS molecules and affect the coordination complex between Ca2/ and PS. In fact, we have recently found that a-tocopherol produced a decrease in the the number of binding sites and a reduction in the apparent affinity for Ca2/ by dimyristoylphosphatidylserine (40). The second factor is the reduced ‘‘fluidity’’ or increase in order of membranes containing a-tocopherol, as measured by fluorescence polarization (41), electron spin resonance (16) and FT-IR spectroscopy (11), which implies a reduced mobility. Membranes in the fluid state are considerably more prone to fusion than those in the solid phase (42). The reported decrease in the fluidity of the membrane by a-tocopherol may contribute to the lower fusion rate and release of contents. In this report a-tocopherol behaves like cholesterol, which has been shown to reduce the ‘‘fluidity’’ of membranes (43) and also to similarly decrease the overall rate of Ba2/- and Ca2/-induced fusion in PS vesicles (44, 45). However, clear differences can be observed between cholesterol and a-tocopherol: first, the observation that cholesterol, as opposed to a-tocopherol, did not reduce the leakage of internal contents during fusion (45), and second, that the effects of cholesterol are observed at considerably higher concentration (ú20 mol %) than those reported here for a-tocopherol. In addition to that, the effect of cholesterol seems to be somehow more complex, since under certain conditions it can also increase the rate of fusion (44, 45). It is interesting to note that a-tocopherol has been reported to preferentially partition into the most fluid domain when incorporated into mixed phospholipid vesicles (12). This was considered to be important because it would cause a-tocopherol to be associated to more unsaturated fatty acyl chains, hence facilitating its peroxidative-protecting task. Considering the results shown here, this location of a-tocopherol would also be important because these more fluid areas are precisely those more prone to undergo fusion and hence would facilitate its possible role as a membrane stabilizer against fusion. A third factor is the possibility of the increased hydration of the membrane surface with increasing atocopherol. Studies on PC/a-tocopherol vesicles by FTIR spectroscopy indicated that the presence of a-tocopherol produced a shift of the C|O stretching mode
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to lower wavenumbers (11); this suggests an increase in the proportion of carbonyl groups of the lipid which are in a more hydrated form. This hypothetically higher hydration would make more difficult a complete dehydration by Ca2/ and the formation of packing defects. This effect of a-tocopherol on PS bilayers which leads to a stabilization of the membrane against fusion may have the same origin as other effects described for the vitamin on membranes. In this respect, a-tocopherol has been shown to inhibit protein kinase C activity (46) which is known to require PS for activation. Inversely, 1,2-dioleoylglycerol, which is a well known activator of protein kinase C (47), has been shown to stimulate Ca2/-induced PS vesicle fusion (48, 49). It might be suggested that the perturbation exerted by a-tocopherol on PS molecules, which is responsible for the stabilization of membrane against fusion, could also affect other roles of PS in membrane function. We have described, in this context, that a-tocopherol decreases the affinity of PS for 45Ca2/ (40), stressing again that this vitamin molecule perturbs the lipid–water interface of the membrane, reducing the capacity of PS to interact with Ca2/ and participate in membrane fusion. In summary, a-tocopherol has been shown to produce a decrease in the rate of fusion and to inhibit the release of contents of Ca2/-induced PS vesicle fusion. This work reinforces the notion that a-tocopherol has the important capacity of acting as membrane stabilizer in addition to its well known function as a natural antioxidant.
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10. Lai, M. Z., Du¨zgu¨nez, N., and Szoka, F. C. (1985) Biochemistry 24, 1646–1653. 11. Villalaı´n, J., Aranda, F. J., and Go´mez-Ferna´ndez, J. C. (1986) Eur. J. Biochem. 158, 141–147. 12. Ortiz, A., Aranda, F. J., and Go´mez-Ferna´ndez, J. C. (1987) Biochim. Biophys. Acta 898, 214–222. 13. Wassall, S. R., Thevalt, J. L., Wong, L., Gorrisen, H., and Cushley, R. J. (1986) Biochemistry 25, 319–326. 14. Srivastasva, S., Phadke, R. S., Govil, G., and Rao, C. N. R. (1983) Biochim. Biophys. Acta 734, 353–362. 15. Severcan, F., and Cannistraro, S. (1988) Chem. Phys. Lipids 47, 129–133. 16. Wassall, S. R., Wang, L., McCabe, R. C. Y., Ehringer, W. D., and Stillwell, W. (1991) Chem. Phys. Lipids 60, 29–37. 17. Aranda, F. J., Coutinho, A., Berberan-Santos, M. N., Prieto, M. J. E., and Go´mez-Ferna´ndez, J. C. (1989) Biochim. Biophys. Acta 985, 26–32. 18. Go´mez-Ferna´ndez, J. C., Villalaı´n, J., Aranda, F. J., Ortiz, A., Micol, V., Coutinho, A., Berberan-Santos, M. N., and Prieto, M. J. E. (1989) Ann. N.Y. Acad. Sci. 570, 109–120. 19. Fukuzawa, K., Ikebata, W., Shibata, A., Kumadari, I., Sakanaka, T., and Urano, S. (1992) Chem. Phys. Lipids 63, 69–75. 20. Ekiel, I. H. J., Hughes, L., Burton, G. W., Iovall, P. A., Ingold, K. H., and Smith, I. C. P. (1988) Biochemistry 27, 1432–1440. 21. Urano, S., Matsuo, M., Sakanaka, T., Uemura, I., Koyama, W., Kumadari, I., and Fukuzawa, K. (1993) Arch. Biochem. Biophys. 303, 10–14. 22. Go´mez-Ferna´ndez, J. C., Villalaı´n, J., and Aranda, F. J. (1993) in Vitamin E in Health and Disease (Packer, L., and Fuchs, J., Eds.), pp. 223–234, Dekker, New York. 23. Salgado, J., Villalaı´n, J., and Go´mez-Ferna´ndez, J. C. (1993) Eur. J. Biophys. 22, 151–155. 24. Micol, V., Aranda, F. J., Villalaı´n, J., and Go´mez-Ferna´ndez, J. C. (1990) Biochim. Biophys. Acta 1022, 194–202. 25. Poste, G., and Nicolson, G. S. (Eds.) (1978) Membrane Fusion: Cell Surface Reviews, Vol. 5, pp. 1–862, Elsevier/North Holland, New York.
ACKNOWLEDGMENTS This work was supported by Grants PB 95/08 from Comisio´n Interministerial de Ciencia y Tecnologı´a, Spain, and PCT 93/22 from Direccio´n General de Educacio´n y Universidad, Comunidad Auto´noma de Murcia, Spain. F.J.A. thanks Dr. A. Ortiz for valuable discussion on lipid peroxidation.
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