The effect of vitamin E on the structure of membrane lipid assemblies
2003; Elsevier BV; Volume: 44; Issue: 10 Linguagem: Inglês
10.1194/jlr.m300146-jlr200
ISSN1539-7262
AutoresA. L. Bradford, Jeffrey Atkinson, Nola Fuller, R.P. Rand,
Tópico(s)Lipid metabolism and biosynthesis
ResumoThe effects of vitamin E on the activity of membrane-dependent enzymes suggest that it acts indirectly by modifying some properties of the lipid host. The effects of α-tocopherol (α-T) and α-tocopherol hemisuccinate (α-THS) on phospholipid monolayer structure, curvature, and bending elasticity were examined using X-ray diffraction and the osmotic stress method. These ligands were mixed with the hexagonal phase-forming lipid, dioleoylphosphatidylethanolamine (DOPE). Increasing levels up to 50 mol% α-T in DOPE in excess water result in a systematic decrease in the lattice dimension. Analysis of the structural changes imposed by α-T shows that it contributes a spontaneous radius of curvature of −13.7 Å. This unusually negative value is comparable to diacylglycerols. α-T does not affect the bending elasticity of these monolayers. α-THS in its charged form decreases membrane curvature, but in its undissociated neutral form has a qualitatively similar but reduced effect on monolayer curvature, as does α-T.We discuss these results in terms of the local stresses such ligands would produce in the vicinity of a membrane protein, and how one might expect proteins to respond to such stress. The effects of vitamin E on the activity of membrane-dependent enzymes suggest that it acts indirectly by modifying some properties of the lipid host. The effects of α-tocopherol (α-T) and α-tocopherol hemisuccinate (α-THS) on phospholipid monolayer structure, curvature, and bending elasticity were examined using X-ray diffraction and the osmotic stress method. These ligands were mixed with the hexagonal phase-forming lipid, dioleoylphosphatidylethanolamine (DOPE). Increasing levels up to 50 mol% α-T in DOPE in excess water result in a systematic decrease in the lattice dimension. Analysis of the structural changes imposed by α-T shows that it contributes a spontaneous radius of curvature of −13.7 Å. This unusually negative value is comparable to diacylglycerols. α-T does not affect the bending elasticity of these monolayers. α-THS in its charged form decreases membrane curvature, but in its undissociated neutral form has a qualitatively similar but reduced effect on monolayer curvature, as does α-T. We discuss these results in terms of the local stresses such ligands would produce in the vicinity of a membrane protein, and how one might expect proteins to respond to such stress. α-Tocopherol (α-T) has been shown to have two major roles in membranes since it was first discovered 1) as a lipid-soluble antioxidant that acts to prevent free radical damage of polyunsaturated fatty acids (1Bisby R.H. Interactions of vitamin E with free radicals and membranes.Free Radic. Res. Commun. 1990; 8: 299-306Google Scholar, 2Burton G.W. Foster D.O. Perly B. Slater T.F. Smith I.C.P. Ingold K.U. Biological antioxidants.Philos. Trans. R. Soc. Lond. Biol. Sci. 1985; 311: 565-578Google Scholar, 3Srivastava S. Phadke R.S. Govil G. Rao C.N.R. Fluidity, permeability and antioxidant behaviour of model membranes incorporated with alpha-tocopherol and vitamin E acetate.Biochim. Biophys. Acta. 1983; 734: 353-362Google Scholar, 4Burton G.W. Ingold K.U. Vitamin E: applications of the principles of physical organic chemistry to the exploration of its structure and function.Acc. Chem. Res. 1986; 19: 194-201Google Scholar, 5Burton G.W. Ingold K.U. Vitamin E as an in vitro and in vivo antioxidant.Ann. N. Y. Acad. Sci. 1989; 570: 7-22Google Scholar), and 2) as a membrane-stabilizing agent through its van der Waals interaction with membrane phospholipids. This latter ability to stabilize membranes may help to prevent the damaging actions of phospholipases, although this is still under debate (6Kagan V. Tocopherol stabilizes membrane against phospholipase A, free fatty acids, and lysophospholipids.Ann. N. Y. Acad. Sci. 1989; 570: 121-135Google Scholar, 7Salgado J. Villalian J. Gomez-Fernandez J.C. Alpha-tocopherol interacts with natural micelle-forming single-chain phospholipids stabilizing the bilayer phase.Arch. Biochem. Biophys. 1993; 306: 368-376Google Scholar, 8Wang X. Quinn P.J. Vitamin E and its function in membranes.Prog. Lipid Res. 1999; 38: 309-336Google Scholar, 9Erin A.N. Gorbunov N.V. Brusovanik V.I. Tyurin V.A. Prilipko L.L. Stabilization of synaptic membranes by alpha-tocopherol against the damaging action of phospholipases. Possible mechanism of biological action of vitamin E.Brain Res. 1986; 398: 85-90Google Scholar, 10Chandra V. Jasti J. Kaur P. Betzel C. Srinivasan A. Singh T.P. First structural evidence of a specific inhibition of phospholipase A(2) by alpha-tocopherol (Vitamin E) and its implications in inflammation: crystal structure of the complex formed between phospholipase A(2) and alpha-tocopherol at 1.8 A resolution.J. Mol. Biol. 2002; 320: 215-222Google Scholar). α-T has also been shown to inhibit protein kinase C (11Azzi A. Stocker A. Vitamin E: non-antioxidant roles.Prog. Lipid Res. 2000; 39: 231-255Google Scholar, 12Azzi A. Breyer I. Feher M. Pastori M. Ricciarelli R. Spycher S. Staffieri M. Stocker A. Zimmer S. Zingg J.M. Specific cellular responses to alpha-tocopherol.J. Nutr. 2000; 130: 1649-1652Google Scholar, 13Tasinato A. Boscoboinik D. Bartoli D. Maroni P. Azzi A. d-alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.Proc. Natl. Acad. Sci. USA. 1992; 92: 12190-12194Google Scholar), apparently without directly binding to the enzyme but rather by activating the translocation of the protein phosphatase 2A to the plasma membrane (14Ricciarelli R. Azzi A. Regulation of recombinant PKC alpha activity by protein phosphatase 1 and protein phosphatase 2A.Arch. Biochem. Biophys. 1998; 355: 197-200Google Scholar). These and other observations, such as those of the effect of α-T on diacylglycerol (DAG) kinase (15Koya D. Lee I.K. Ishii H. Kanoh H. King G.L. Prevention of glomerular dysfunction in diabetic rats by treatment with d-alpha-tocopherol.J. Am. Soc. Nephrol. 1997; 8: 426-435Google Scholar, 16Tran K. Proulx P.R. Chan A.C. Vitamin E suppresses diacylglycerol (DAG) level in thrombin-stimulated endothelial cells through an increase of DAG kinase activity.Biochim. Biophys. Acta. 1994; 1212: 193-202Google Scholar), CoA-independent transacylase (17Tran K. D'Angelo A.F. Choy P.C. Chan A.C. Vitamin E enhances the acylation of 1-O-alkyl-sn-glycero-3-phosphocholine in human endothelial cells.Biochem. J. 1994; 298: 115-119Google Scholar), and phospholipase D (18Yamamoto I. Konto A. Handa T. Miyajima K. Regulation of phospholipase K activity by neutral lipids in egg-yolk phosphatidylcholine small unilamellar vesicles and by calcium ion in aqueous medium.Biochim. Biophys. Acta. 1995; 1233: 21-26Google Scholar) suggest that the modulation of enzyme activity may have more to do with the effect tocopherol has on membrane structure, particularly inasmuch as all these enzymes act on substrates that are in membranes or are activated/inhibited when translocated to a membrane surface.α-Tocopherol hemisuccinate (α-THS) also exhibits biological activity and has been implicated as a cancer chemopreventative agent with chemotherapeutic potential (19Yu W. Isreal K. Liao Q.Y. Aldaz C.M. Sanders B.G. Kline K. Vitamin E succinate (VES) induces Fas sensitivity in human breast cancer cells: role for Mr 43,000 Fas in VES-triggered apoptosis.Cancer Res. 1999; 59: 953-961Google Scholar). It has been shown that this succinate derivative can induce apoptosis through a variety of pathways, such as G1 cell blockage, DNA synthesis arrest, and activation of transforming growth factor β and enhanced expression of its type II receptor (20Kline K. Yu W. Sanders B.G. Vitamin E: mechanisms of action as tumor cell growth inhibitors.in: Prasad K.N. Cole W.C. Cancer and Nutrition. IOS Press, Amsterdam1998: 37-53Google Scholar). α-THS differs from the parent molecule by having a succinic acid moiety esterified to the chroman phenol, and it is this modification that eliminates the classical antioxidant activity of α-T. A related ester, cholesterol hemisuccinate, is known to interact with membrane components, reducing acyl chain mobility and increasing the surface charge (21Massey J.B. Effect of cholesteryl hemisuccinate on the interfacial properties of phosphatidylcholine bilayers.Biochim. Biophys. Acta. 1998; 1415: 193-204Google Scholar).Because α-T and α-THS may alter enzyme activity by changing the biophysical properties of the membrane, it is of interest to determine what effect the addition of these compounds has on the structure of lipid assemblies. To investigate the effect of α-T and α-THS on such assemblies, we report here the use of X-ray diffraction to characterize the structures formed by hydrated lipid membranes (22Chen Z. Rand R.P. The influence of cholesterol and phospholipid membrane curvature and bending elasticity.Biophys. J. 1997; 73: 267-276Google Scholar). Hubner et al. (23Hubner S. Couvillon A.D. Kas J.A. Bankaitis V.A. Vegners R. Carpenter C.L. Janmey P.A. Enhancement of phosphoinositide 3-kinase (PI 3-kinase) activity by membrane curvature and inositol-phospholipid-binding peptides.Eur. J. Biochem. 1998; 258: 846-853Google Scholar) and others (24Drobnies A.E. Davies S.M. Kraayenhof R. Epand R.F. Cornell R.B. CTP:phosphocholine cytidylytransferase and protein kinase C recognize different physical features of membranes: differential responses to an oxidized phosphatidylcholine.Biochim. Biophys. Acta. 2002; 1564: 82-90Google Scholar, 25Davies S.M. Epand R.F. Kraayenhof R. Cornell R.B. Regulation of CTP:phosphocholine cytidylytransferase activity by the physical properties of lipid membranes: an important role for stored curvature strain energy.Biochemistry. 2001; 40: 10522-10531Google Scholar, 26Cornell R.B. Regulation of CTP: phosphocholine cytidylytransferase by lipids. 2. Surface curvature, acyl chain length, and lipid-phase dependence for activation.Biochemistry. 1991; 30: 5881-5888Google Scholar) have shown that curvature affects the activity of membrane enzymes in vesicles. We have measured the influence of α-T and α-THS on the spontaneous curvature and bending modulus (Kcp) when added to dioleoylphosphatidylethanolamine (DOPE) lipid monolayers. Among the multitude of heterogeneous membrane sites, such model systems as explored here are intended to reflect only the properties of specific membrane sites that contain α-T.MATERIALS AND METHODSSample preparationα-T was obtained by hydrolysis (MeOH, K2CO3) of commercially available natural source α-tocopheryl acetate and was purified by silica gel column chromatography before use. α-THS was obtained from Sigma Aldrich Canada (Oakville, Ontario) and was used without further purification. The sodium salt of α-THS was prepared by titration of a diethyl ether solution of a α-THS with NaOH in EtOH. The resulting precipitate was rinsed with ether-EtOH (20:1; v/v) and dried in vacuo. Synthetic DOPE was purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. DOPE and α-T were stored under nitrogen at −18°C. α-THS was stored at room temperature, and all water used in experiments was double distilled.The desired lipid mixtures were produced by combining the required amounts of DOPE with either α-T or α-THS in chloroform solution. The chloroform was then removed by rotary evaporation under a constant stream of nitrogen, followed by vacuum desiccation. The dry lipid mixtures were hydrated to varying degrees by gravimetric addition of double-distilled water or excess amounts of polyethyleneglycol solutions of different measured osmotic pressures. The hydrated samples were then given another 72 h to equilibrate at room temperature. The equilibrated samples were then mounted between two mica windows with powdered teflon (as an X-ray calibration standard, repeat spacing of 4.87 Å) and examined by X-ray diffraction.X-ray diffractionTo characterize the structures formed by the final hydrated lipid mixture, an X-ray diffraction method was used. A Rigaku rotating anode generator produced a CuKα1 line (λ = 1.540 Å), which was isolated using a bent quartz crystal monochromator. The diffraction patterns of the hydrated lipid were photographed using Guinier X-ray cameras. The temperature of the sample was controlled by thermoelectric elements and maintained at 22°C ± 0.5°C. Samples that formed hexagonal phases (HIIs) are characterized by at least three X-ray spacings with the ratios to the dimension of the first order [hexagonal lattice dimension (dhex)] of 1, 1/√3, 1/√4, 1/√7, 1/√9, 1/√12, etc. Lattice dimensions of the hexagonal structures could be measured to ±0.1 Å on any one sample.Structural analysisHIIs are two-dimensional hexagonal lattices with the water cores centered on the prism axes and lined with lipid polar groups, while the rest of the lattice is filled with the hydrocarbon chains. For an HII of known composition, its lattice can be divided into compartments, as shown in Fig. 1, each containing the volume fractions of lipid and water. This average division follows the method originally introduced by Luzzati and Husson (27Luzzati V. Husson F. X-ray diffraction studies of lipid-water systems.Cell Mol. Biol. 1962; 12: 207-219Google Scholar) and depends only on knowledge of the specific volumes of the molecular components and their linear addition. Some of the physicochemical and structural parameters for the lipid components used in this study are listed in Table 1.TABLE 1Physiochemical and structural parameters of molecules used in this studyMolecular MassDensitySpecific Volumeg/cm3cm3/gDioleoylphosphatidylethanolamine744.0 1.001.00α-T430.7 0.951.05α-THS530.8 0.95aDensity of α-THS derivative is an approximation based on the density of α-T acetate.1.05α-T, α-tocopherol; α-THS, α-tocopherol hemisuccinate.a Density of α-THS derivative is an approximation based on the density of α-T acetate. Open table in a new tab The water and lipid components can be separated through the introduction of an idealized cylindrical interface, the Luzzati surface, in which all of the water is inside this cylinder and all of the lipid is outside. The radius of this water cylinder, Rw, is related to both the volume fraction of the water in the sample, ϕw, and dhex, as follows in equation 1.Rw=dhex2ϕwπ3(Eq. 1) The area per lipid molecule at the Luzzati surface is given in equation 2 as: Aw=2ϕwV1(1−ϕw)Rw(Eq. 2) where V1 is the volume of the lipid molecule. ϕw is calculated from the weight fraction of water using the specific volumes stated in Table 1.When the volume of a lipid molecule is used it is based on the notion of an effective molecule, i.e., one phospholipid, DOPE of volume Vpl, plus x-(α-T) molecules, where Vα-T is the volume of α-T and x is the molar ratio of α-T to phospholipids. The effective molecular volume is then given in equation 3 as: V1=Vp1+xVα−T(Eq. 3) The molecular area (A) at, and the radius (R) of, any cylindrical dividing surface separated from the Luzzati surface by volume (V), are given in equations 4 and 5 as: A=Aw1+1−ϕwϕwVV1(Eq. 4) R=Rw1+1−ϕwϕwVV1(Eq. 5) To determine if there is a surface of constant area, i.e., a pivotal plane, equation 4 can be expressed in a form that uses normalized areas and volumes (28Leikin S. Kozlov M.M. Fuller N.L. Rand R.P. Measured effects of diacylglycerol on structural and elastic properties of phospholipid membranes.Biophys. J. 1996; 71: 2623-2632Google Scholar) (equation 6): Aw2V12=Ap2V12−2VpV1(AwV1Rw)(Eq. 6) If a straight line results from plotting (Aw/Vl)2 versus Aw/(VlRw) (our "diagnostic plot"), then the system has a dividing surface that has a constant molecular area and is defined as the pivotal plane. The slope (Vp/Vl) of the diagnostic plot gives the position of the pivotal plane. Equation 5 can then be used to calculate the radii of curvature (Ro) of the HII monolayers. At full hydration, R = Rop, the spontaneous curvature of the lipid monolayers. Once Rop has been determined for the mixed monolayer, the intrinsic curvature of the individual lipids can also be determined using equation 7, if it is linear: 1R0p=(1−mα−T)1R0pDOPE+mα−T1R0pα−T(Eq. 7) where the molar fraction α-T is given by mα−T = x/(1+x) (28Leikin S. Kozlov M.M. Fuller N.L. Rand R.P. Measured effects of diacylglycerol on structural and elastic properties of phospholipid membranes.Biophys. J. 1996; 71: 2623-2632Google Scholar).Once the pivotal plane is known, the elastic free energy, F, of the HII can be approximated by the energy of bending (29Helfrich W. Elastic properties of lipid bilayers: theory and possible experiments.Z. Naturforsch. 1973; 28: 693-703Google Scholar, 30Kirk G.L. Gruner S.M. Stein D.L. A thermodynamic model of the lamellar to inverse hexagonal phase transition of lipid membrane-water systems.Biochemistry. 1984; 23: 1093-1102Google Scholar), as shown in equation 8.F=12KcpAp(1Rp−1R0p)(Eq. 8) where Kcp is the bending modulus, Ap is the molecular area, and Rp and R0p are the Ro and the spontaneous Ro respectively.By comparing the elastic free energy of the lipid mixture (equation 8) under conditions of osmotic stress, with the osmotic work done by the osmotic stress (Π), a plot of (ΠRp2) versus (1/Rp) gives, from the slope, the monolayer Kcp (31Gruner S.M. Parsegian V.A. Rand R.P. Directly measured deformation energy of phospholipid HII hexagonal phases.Faraday Discuss. Chem. Soc. 1986; 81: 29-37Google Scholar, 32Rand R.P. Fuller N.L. Gruner S.M. Parsegian V.A. Membrane curvature, lipid segregation, and structural transitions for phospholipids under dual-solvent stress.Biochemistry. 1990; 29: 76-87Google Scholar), as shown in equation 9.πRp2=2Kcp(1Rp−1R0p) (Eq. 9) RESULTSThe relationship between mol% α-T or α-THS in DOPE and the equilibrium lattice spacing of the resultant HII is shown in Fig. 2. For both α-T and α-THS, the dimension of the HII decreases with increasing tocopherol content. It appears that both α-T and α-THS are increasing the curvature of the mixed monolayers, α-T more so than α-THS.Fig. 2Plot of the equilibrium lattice spacing, hexagonal lattice dimension (dhex), for lipid mixtures of increasing amounts of α-tocopherol (α-T) (square), α-tocopherol hemisuccinate (α-THS) (circle), and α-T sodium-succinate (triangle) in dioleoylphosphatidylethanolamine (DOPE).View Large Image Figure ViewerDownload (PPT)Gravimetric phase diagrams covering the full hydration range for three mixtures of α-T with DOPE are shown in Fig. 3. The dhex is shown as it varies with weight fraction lipid in water. dhex for all the single HIIs increases with water content until a maximum is reached. That maximum depends on the α-T content. Several α-T/DOPE ratios were prepared and their maximally hydrated equilibrium hexagonal dimensions determined (Fig. 3). The weight fraction of lipid at the maximum hydration for every α-T/DOPE ratio was determined from the intercept of the best-fit curve below excess water, with the average maximum dimension in excess water.Fig. 3Lattice dimension, dhex, as a function of water content for the HII formed by mixtures containing DOPE and the indicated mole% α-T: 0% (open circle); 10% (open triangle); 20% (open square); and 25% (open diamond). In a separate experiment (closed symbols), the trend in lattice dimension change with α-T content, and at full hydration was more accurately determined by systematic serial dilution of lipid mixtures: 0% (closed circle); 10% (closed inverted triangle); 20% (closed square); 30% (upright closed triangle); 40% (round ring); 50% (square ring); and 70% (cross).View Large Image Figure ViewerDownload (PPT)Figure 4shows the diagnostic plots of (Aw/Vl)2 versus Aw/(VlRw) for all α-T/DOPE mixtures. The linearity of this relationship indicates that there is a well-defined pivotal plane for all of these mixtures. Therefore, there exists a position in the monolayer that does not change area even as the monolayer is bent upon dehydration. The position of this pivotal plane is given by the slope of the relationship in Fig. 4, (Vp/Vl). Vp/Vl for α-T in DOPE is 0.32.Fig. 4Diagnostic plots for the lipid mixtures: 0% (circle); 10% (triangle); 20% (square); and 25% (diamond) of Fig. 3. Linearity indicates a pivotal plane, the slope of the line gives its position, Vp/Vl, and its intercept, Ap/Vl, gives the effective molecule (see Materials and Methods) at this plane.View Large Image Figure ViewerDownload (PPT)The spontaneous Ro of the lipid monolayers, Rop, is calculated from equations 1 and 5 using the equilibrium volume fraction in excess water, the maximum lattice dimension for dhex, and the value of Vp/Vl. Figure 5shows that α-T increases monolayer curvature. The linear relationship allows the apparent spontaneous curvature of the individual lipids to be determined from equation 7. Rop for α-T is −13.7 Å, making it a membrane component with one of the most negative curvatures measured, in the category of the diacylglycerols, and considerably more negative than cholesterol. R0p for DOPE is −29.4 Å, which is consistent with several previous studies (22Chen Z. Rand R.P. The influence of cholesterol and phospholipid membrane curvature and bending elasticity.Biophys. J. 1997; 73: 267-276Google Scholar, 27Luzzati V. Husson F. X-ray diffraction studies of lipid-water systems.Cell Mol. Biol. 1962; 12: 207-219Google Scholar). These curvature values are shown in Table 2.Fig. 5Plot of spontaneous monolayer curvature, 1/R0p, for DOPE/α-T mixtures as a function of α-T content.View Large Image Figure ViewerDownload (PPT)TABLE 2Comparison of spontaneous radii of curvature and bending moduli for individual lipidsRo (Å)Kc/kTReferencesDOG (in DOPE)−11.528DOG (in DOPC)−10.133DCG (in DOPC)−13.333α-T (DOPE)−13.7DOPE−28.51128DOPE-tetradecane−28.71222Cholesterol (in DOPE)−22.822Cholesterol (in DOPC)−27.2 (32°C)22DCG, dicaprylglycerol; DOG, dioleoylglcerol; DOPC, dioleoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine; Ro, radius of curvature. Open table in a new tab Following equation 9, Fig. 6shows the ΠRp2 versus 1/Rp plots, a measure of how easy it is to bend these monolayers as water is withdrawn osmotically. From the slopes of these plots, we are able to determine the Kcp of the α-T/DOPE mixtures. The Kcp, plotted in Fig. 7, show that α-T does not change the bending elasticity of these monolayers.Fig. 6Plot relating the osmotic work required to dehydrate the HII of DOPE/α-T mixtures with the change in monolayer curvature 1/Rp. The slope, determined by least-squares fit (all r values > 0.96), gives the measure of the bending modulus (Kcp): 15% (inverted triangle); 20% (square); 25% (diamond); and 30% (upright triangle).View Large Image Figure ViewerDownload (PPT)Fig. 7Kcp calculated for increasing amounts of α-T in DOPE mixtures.View Large Image Figure ViewerDownload (PPT)DISCUSSIONWe have investigated the modifying effects of α-T on phospholipid structures with the hope that such measurements may lead to an understanding of the mechanism by which α-T affects the activity of membrane resident enzymes. Inasmuch as direct binding of α-T has not been demonstrated, it is possible that tocopherol(s) modify the physical properties of the protein's lipid milieu. Local curvature is one physical parameter that is thought to produce local stress in bilayer membranes and so affect protein-lipid interactions and, thereby, protein conformation and activity. One clear example is that of PI-3 kinase (23Hubner S. Couvillon A.D. Kas J.A. Bankaitis V.A. Vegners R. Carpenter C.L. Janmey P.A. Enhancement of phosphoinositide 3-kinase (PI 3-kinase) activity by membrane curvature and inositol-phospholipid-binding peptides.Eur. J. Biochem. 1998; 258: 846-853Google Scholar), whose activation appears to depend on the curvature of its host lipid vesicle. We have measured the contribution of several different types of lipid-to-membrane curvature. This is done by determining the stress free, or intrinsic, or spontaneous curvature of lipid assemblies. The higher this curvature, the more local stress a lipid will produce when confined to a flat bilayer membrane, and the higher the driving force in lipid–protein interactions. We have illustrated this hypothetical mechanism in Fig. 8.Fig. 8The conformational changes that a membrane-bound enzyme (A, B, or C, large circles and connected ovals) might undergo to compensate for the addition of lipid with either positive (upright triangle) or negative (inverted triangle) curvature in its vicinity.View Large Image Figure ViewerDownload (PPT)Several enzymes are affected by α-T without direct binding (11Azzi A. Stocker A. Vitamin E: non-antioxidant roles.Prog. Lipid Res. 2000; 39: 231-255Google Scholar, 12Azzi A. Breyer I. Feher M. Pastori M. Ricciarelli R. Spycher S. Staffieri M. Stocker A. Zimmer S. Zingg J.M. Specific cellular responses to alpha-tocopherol.J. Nutr. 2000; 130: 1649-1652Google Scholar, 13Tasinato A. Boscoboinik D. Bartoli D. Maroni P. Azzi A. d-alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.Proc. Natl. Acad. Sci. USA. 1992; 92: 12190-12194Google Scholar), and that may be a result of a general effect tocopherol has on membrane structure. In this study, we have shown that α-T has one of the smallest intrinsic Ros, and therefore contributes one of the highest negative curvatures measured in membranes, comparable to that of the DAGs (33Szule J.A. Fuller N.L. Rand R.P. The effects of acyl chain length and saturation of diacylglycerols and phosphatidylcholines on membrane monolayer curvature.Biophys. J. 2002; 83: 977-984Google Scholar). According to our hypothesis, α-T's action on membrane-resident enzymes is through the local curvature it produces in the vicinity of these proteins. This curvature stress results from the addition of relatively more mass to the hydrocarbon portion of the membrane monolayers than to the polar portion. One might expect that proteins in the presence of α-T would react to this stress. Stress reduction would result from a conformational change involving a movement of protein mass from the hydrocarbon to the polar group region of the monolayer. This change is the example shown schematically in the transition (Fig. 8B–C).α-T does not apparently affect the Kcp of the membrane monolayers. Such Kcp is relevant to any local deformation of the monolayer as might be required by the protein in adjusting to lipid-protein packing of the new conformation.We could not make equivalent measurements for α-THS, but the qualitative effects that it has on local curvature appear clear. α-T sodium succinate increased the hexagonal lattice of DOPE, indicating the addition of a much lower spontaneous curvature than that of α-T, a likely consequence of charge repulsion at the polar group layer. The protonated form of α-THS, however, had the same, although smaller, effect on the DOPE lattice dimension as did α-T. Thus, it behaved as if it were uncharged (undissociated), and its larger polar group likely added less negative curvature than did α-T itself. α-Tocopherol (α-T) has been shown to have two major roles in membranes since it was first discovered 1) as a lipid-soluble antioxidant that acts to prevent free radical damage of polyunsaturated fatty acids (1Bisby R.H. Interactions of vitamin E with free radicals and membranes.Free Radic. Res. Commun. 1990; 8: 299-306Google Scholar, 2Burton G.W. Foster D.O. Perly B. Slater T.F. Smith I.C.P. Ingold K.U. Biological antioxidants.Philos. Trans. R. Soc. Lond. Biol. Sci. 1985; 311: 565-578Google Scholar, 3Srivastava S. Phadke R.S. Govil G. Rao C.N.R. Fluidity, permeability and antioxidant behaviour of model membranes incorporated with alpha-tocopherol and vitamin E acetate.Biochim. Biophys. Acta. 1983; 734: 353-362Google Scholar, 4Burton G.W. Ingold K.U. Vitamin E: applications of the principles of physical organic chemistry to the exploration of its structure and function.Acc. Chem. Res. 1986; 19: 194-201Google Scholar, 5Burton G.W. Ingold K.U. Vitamin E as an in vitro and in vivo antioxidant.Ann. N. Y. Acad. Sci. 1989; 570: 7-22Google Scholar), and 2) as a membrane-stabilizing agent through its van der Waals interaction with membrane phospholipids. This latter ability to stabilize membranes may help to prevent the damaging actions of phospholipases, although this is still under debate (6Kagan V. Tocopherol stabilizes membrane against phospholipase A, free fatty acids, and lysophospholipids.Ann. N. Y. Acad. Sci. 1989; 570: 121-135Google Scholar, 7Salgado J. Villalian J. Gomez-Fernandez J.C. Alpha-tocopherol interacts with natural micelle-forming single-chain phospholipids stabilizing the bilayer phase.Arch. Biochem. Biophys. 1993; 306: 368-376Google Scholar, 8Wang X. Quinn P.J. Vitamin E and its function in membranes.Prog. Lipid Res. 1999; 38: 309-336Google Scholar, 9Erin A.N. Gorbunov N.V. Brusovanik V.I. Tyurin V.A. Prilipko L.L. Stabilization of synaptic membranes by alpha-tocopherol against the damaging action of phospholipases. Possible me
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