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Phosphatidylcholine and cholesterol interactions in model membranes
Chemistry and Physics of Lipids, 33 (1983) 313-322 Elsevier Scientific Publishers Ireland Ltd.
313
PHOSPHATIDYLCHOLINE AND CHOLESTEROL INTERACTIONS IN MODEL MEMBRANES
W. G U Y E R and K. B L O C H
James Bryant Conant Laboratories, Department of Chqmistry, Harvard University, Cambridge, MA 02138 (U.S.A.) Received D e c e m b e r 8th, 1982
accepted April 30th, 1983
Various phosphatidylcholines differing either in the stereochemistry around their chiral center or in the position of a cis double bond along the acyl chains were synthesized in order to study critical contact regions in the phospholipid molecule with adjacent cholesterol in model m e m b r a n e s . Microviscosities calculated from fluorescence depolarization of diphenylhexatriene and chain order from spin label studies were m e a s u r e d to monitor physical m e m b r a n e properties. T h e enhancing effect of cholesterol on the microviscosity of m e m b r a n e s containing phosphatidylcholines with comparable acyl chain length was largest when the two acyl chains were saturated and smallest when both were unsaturated. M e m b r a n e s prepared from phosphatidylcholines having a single cis double bond at different positions along the sn-2 acyl chain showed roughly the same changes of microviscosity or chain order upon incorporation of cholesterol. No discrimination was evident in the interaction between cholesterol and enantiomeric phosphatidylcholines or between the enantiomeric phosphatidylcholine molecules themselves. We conclude that the rigidifying effect of cholesterol in m e m b r a n e s does not depend on specific sites of interaction and that with respect to physical m e m b r a n e properties phosphatidylcholine behaves as an achiral molecule.
Keywords: lipid-lipid interaction; phosphatidylcholine; cholesterol; structural specificity; chiral recognition.
Introduction Sterols and phospholipids are the major lipids of cellular membranes, and it appears that for cell growth and function, a balanced ratio of the two components has to be maintained. At the physiological or biochemical level the function of sterols in membranes is not well understood. However, experimentally induced changes in the sterol concentration have been shown to alter the physical state of the membrane and in turn modulate a broad range of cellular processes [1-4]. Such changes of physical state are often accompanied by adjustments in the structure of the phospholipid molecule [5-7]. Various physiological responses are believed to be related in some manner to membrane fluidity which in turn is controlled by the dynamic 0009-3084/83/$03.00 O 1983 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
314 interactions between the various lipid components in the hydrophilic polar zone and the hydrophobic core. The structure of cholesterol appears to be especially well designed for modulating the physical state of phospholipid bilayers [8, 9]. Evidence based on a variety of physical techniques has been interpreted as showing that cholesterol entering the membrane aligns in parallel with the extended acyl chains of the phospholipids, the sterol hydroxyl group pointing toward the water-bilayer interface [10]. Since both sterol and phospholipid are chiral but only one enantiomer of each is commonly found in nature, stereochemical factors could conceivably be of importance for the strength of interaction between the two lipid components. Some current, more explicit membrane models specify distinct regions of interactions, namely (a) hydrogen bonding between the sterol hydroxyl function and some polar element of the phospholipid head group, e.g. the ester carbonyl [1 1, 12] and (b) hydrophobic interactions in the core of the bilayer which are postulated to be sensitive to the number and location of double bonds in the phospholipid acyl chains [12]. In the present study we describe rigidifying effects of cholesterol in membrane bilayers prepared from various synthetic positional isomers and enantiomers of phosphatidylcholine. These phosphatidylcholines were designed to detect perturbations in the interactions involving either the polar or the hydrophobic contact regions of cholesterol.
Materials and methods
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (16 : 0/16:0-sn3-PC), t,2dipalmitoyl-rac-glycero-3-phosphocholine (16:0/16:0-rac-PC) and 1,2dioleoyl-sn-glycero-3-phosphocholine (18: lc9/18: lc9-pc) were purchased from Sigma Chemical Co. 2,3-Dipalmitoyl-sn-glycero- 1-phosphocholine (16 : 0/16 : O-sn 1-PC) was prepared from 16 :0/16 :0-rac-PC by enzymatic hydrolysis of the sn-3 enantiomer with phospholipase A2 (lyophilized Crotalus adamanteus venom from Sigma Chemical Co.) [13], leaving the sn-1 enantiomer intact. The resulting crude lipid mixture was taken to dryness by rotary evaporation and the sn-1 enantiomer purified by chromatography on silica gel (Unisil Clarkson Chemical Co) (100g/g lipid) with chloroform/methanol as solvent. The phospholipid fraction was passed through a column of Rexyn 1-300 mixed bed ion exchanger (Fisher Scientific Co.) (50 ml/mmol phosphatidylcholine) with chloroform/methanol/water (4 : 5 : 1, by vol.) as the solvent system. This step removed any possible contamination with Ca 2+ remaining from the phospholipase A2 treatment. The phospholipid was rechromatographed on a silica gel column as above and finally crystallized from acetone/chloroform.
315
1-Palmitoyl-2-petroselinoyl-sn-glycero-3-phosphocholine (16:0/18 : lc 6PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0/18 : lcg-PC) and 1-palmitoyl-2-vaccenoyl-sn-glycero-3-phosphocholine (16:0/18 : lc11-PC) were synthesized by enzymatic removal of the sn-2 chain from 16:0/16:0sn3-PC with phospholipase A2 [13] and reacylation with the appropriate fatty acid anhydrides under conditions that minimize acyl chain migration [14[. Petroselinic, oleic and vaccenic acids were from Sigma Chemical Co. (nominal purity > 9 8 % ) and the various anhydrides were prepared [15]. All phospholipids were purified by chromatography on Rexyn 1-300 and silica gel and crystallized as outlined above. In order to prevent air oxidation, all steps were carried out under nitrogen. A trace of hydroquinone was added to the solvents throughout synthesis and purification, except for the final crystallization step. The pure phospholipids were stored under argon at -2{}°C. The chemical purity of the phospholipids was checked by TLC. Samples of {}.4 mg of the phospholipids were applied to TLC plates (Si250, J.T. Baker Chemical Co.) and developed with the solvent systems CHCI3/CH3OH/ NH4OHaq (65:28:4, by vol.) and CHCI3/CH3OH/CH3COOH/H20 (50:25:7:3, by vol.) respectively over a distance of 18 cm. Only single spots could be detected upon exposure to iodine or after charring with sulfuric acid. Greater than 98% of the applied sample was recovered from the phospholipid zone as determined by phosphate analysis. The optical purity of 16:0/16:0-PC was verified by measuring rotations with a Perkin Elmer 141 polarimeter. 16:0/16:0-sn3-PC, [a]~ = + 6 . 3 __~_0 . 2 o , 16:0/16:(}-snl-PC [c~]~ = -6.4+_0.2°; (c = 1.6, CHCI3/CH3OH, 1:1). 16:0/16:{}-rac-PC exhibited no optical rotation. Previously reported values for 16:(}/16:0-sn3-PC range from [a]~=+7.{} ° ( c = 5 . 6 , CHC13) [16] to [a]~ = +6.6 ° (c = 8, CHCI3/CH3OH, 1" 1) [t7]. Cholesterol (Aldrich Chemical Co.) was recrystallized twice from 95% ethanol and acetone and dried in vacuo. The fluorescent probe, 1,6diphenyl-l,3,5-hexatriene (DPH) and the spin-labeled fatty acid 5-doxylstearic acid were purchased from Aldrich Chemical Co. and Syva Associates, respectively, and used as supplied. Preparation of liposomes The required amounts of lipids were dissolved in CHCI3/CH3OH (2: 1, by vol.) and the mixture was dried under a stream of nitrogen. Residual solvent was removed in vacuo in a desiccator containing K O H pellets and P20~. For microviscosity measurements of multilamellar dispersions, the aqueous medium (50 mM NaCI, 10 mM sodium phosphate buffer (pH 7.2), 1 mM E D T A ) was supplemented with 10% sucrose, added to the lipid film and the lipids dispersed by shaking on a Vortex mixer for 5 rain above the transition
316 t e m p e r a t u r e (To) of the phospholipid. These dispersions were equilibrated for at least 3 h at the t e m p e r a t u r e chosen for subsequent experiments. Unilamellar vesicles were prepared by sonication with a Heat Systems sonifier Model W-350 (at 20 kHz, 70 W output power, 50% duty cycle) for 15 rain in a conical glass tube. The tip of the soniprobe was immersed to about half of the dispersion height. During sonication, the tube was kept in a water bath above T,. and a stream of H20-saturated nitrogen was passed over the dispersion. No degradation of the lipids could be observed by TLC. Titanium from the soniprobe was removed from the sample by centrifugation for 10 min at 2000 × g and the clear supernatant was kept above T,. and used the same day. Vesicle yield was typically better than 85% as determined by sizing on a column of Bio-Gel A-50m (Bio-Rad Laboratories). Microviscosity measurements Microviscosities were determined in an Elscint microviscosimeter Model MV-1A, with D P H as the fluorescent probe. To 2.5 ml of the lipid suspension (1.25 p~mol total lipid) was added 1 ~1 D P H (1 mM in tetrahydrofuran) with vigorous shaking. This mixture was incubated at 37°C (47°C for 16:0/16 : 0-PC) for 60 min. Fluorescence depolarization was measured at the temperatures indicated under results, and microviscosities calculated according to Shinitzky and Inbar [18]. Electron paramagnetic resonance E P R spectra were recorded at the indicated temperatures on a Varian E-line 9.5 G H z spectrometer equipped with a Varian t e m p e r a t u r e controller. The microwave power was held constant at 20 m W and the modulation amplitude at 0.8 G. Aliquots of 50 ~1 multilamellar lipid dispersions prepared from 2 ~mol total lipid were placed in a 1 m m glass capillary and E P R spectra were recorded. Spin-labeled fatty acids were introduced into the liposomes at a molar ratio of 1:200. 2T~1and 27"1 were derived from the spectra by measuring the splittings between the outer and the inner hyperfine peaks and the order p a r a m e t e r (S) was calculated from these values according to Hubbell and McConneli [19].
Results
Microviscosity values derived from the fluorescence depolarization of D P H provide valid, comparative measures of m e m b r a n e 'fluidity' as a function of m e m b r a n e composition. Figures 1A and B illustrate the effect of
317 TEMPERATURE 50 20
]
40 i
I
30 i
I
20 q
I
i
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5
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(°C) ~
a.
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32
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I
1
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35
36
32
33
3.4
3.5
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3
(°K-l)
1/T.10
Fig. 1. Effect of temperature on the microviscosity(-q) of multilamellar dispersions of phosphatidylcholines (open symbols) and their 1 : 1 mixtures with cholesterol (closed symbols). Averaged data of two to four independent experiments. (a) 18: lc9/18: lc9-pc (©, O); 16:0/16:0-sn3-PC (A, &); 16:0/16:0-rac-PC ([], i ) ; 16:0/16:0-snl-PC (V, V). (b) 16:0/18:1c6-pc (A, &); 16:0/18: lc9-pc ([2, I ) ; 16:0/18: lclLPC (~', V).
t e m p e r a t u r e on the microviscosity of multilamellar lipid dispersions prepared from the synthetic phosphatidylcholines and their 1:1 mixtures with cholesterol. The data are presented as Arrhenius plots. In the t e m p e r a t u r e range studied all phosphatidylcholine-cholesterol mixtures and three of the unsaturated pure phosphatidylcholines show the expected linear relation. In both phospholipids alone and in their 1:1 mixtures with cholesterol, the presence of an unsaturated phospholipid acyl chain causes a decrease of microviscosity. A m o n g the 'mixed' phospholipids, 16:0/18: lc9-pc containing the double bond in the central position along the acyl chain yields the most fluid bilayer. In the absence of cholesterol abrupt changes in slope are observed for the three optically different species of 16:0/16:0-PC (Fig. 1A) and for 16:0/18: lc6-pc (Fig. 1B) attributable to the gel to liquid-crystalline phase transition [20]. The important result is that the curves for the two enantiomers of 16:0/16:0-PC and their racemic mixture are indistinguishable from each other and that all three show a transition t e m p e r a t u r e (midpoint of the change) of Tc = 40.8°C (range 39.3-42.4°C) (Fig. 1A) in good agreement with published values for 1 6 : 0 / 1 6 : 0 - s n 3 - P C [20-22]. For the 16:0/18: Ic6-pc, not previously reported, T~ was 19.4°C (range 18.521.3°C) (Fig. 1B). This value is high c o m p a r e d to those for 16:0/18: lc9-pc and 16:0/18 : lclS-pc, which show a phase transition at a t e m p e r a t u r e lower than the range chosen. The published value for 16:0/18: lc9-pc is Tc < 5 ° C
318
[231. The 'smoothing' effect of cholesterol on the phase transition evident in Figs. 1A and 1B has been observed repeatedly with various methods. Qualitatively similar curves were obtained with unilamellar vesicles (data not shown). Incorporation of increasing amounts of cholesterol into vesicles progressively raises m e m b r a n e microviscosities above Tc [24]. Figure 2 shows the microviscosity increase caused by incremental quantities of cholesterol in unilamellar vesicles prepared from the various phosphatidylcholines. Since the intrinsic microviscosities of the pure phospholipids were different, relative microviscosities were calculated. This presentation of the data seems justified by the finding that microviscosities measured in phosphatidylcholine-cholesterol bilayers by the D P H method obey the Batschinsky relation, which inversely links microviscosity to the free volume in the lipid [25]. Cholesterol raises the microviscosity of 16:0/16:0-PC markedly producing 11 times the value shown by the pure phospholipid at a content of 5[) m o l % . H o w e v e r the responses of the three optically different forms of 16:0/16:0-PC were identical as shown by the three superimposable curves in Fig. 2. The effect of cholesterol on the unsaturated phosphatidylcholines was in general less marked, the di-unsaturated 18:1c9/18 : lcg-PC being least affected. As for the phosphatidylcholines with one unsaturated acyt chain in the sn-2 position, a shift of the central double bond in either direction along the chain only slightly altered the susceptibility to cholesterol. Qualitatively similar results were obtained with multilamellar lipid dispersions with even
TABLE 1 O R D E R P A R A M E T E R S (S) O F P H O S P H O L I P I D A N D P H O S P H O L I P I D CHOLESTEROL MULTILAMELLAR DISPERSIONS The estimated error of $ is +(I.()1. Temperature, 47°(7 for 16:0/16:0-PC, 37°C for all other phospholipids. Cholest erol/phospholipid
16:0/16:0-sn3-PC 16:0/16:0-rac-PC 16 : 0/16 : 0-sn 1-PC
0
1:4
1:2
[).571 0.569 0.565
0.625 0.619 (I.625
0.665 (I.658 [I.665
16 : 0 / 1 8 : l c ~ - P C 16:0/18: Icg-PC 16:0/18: IcLLPC
0.596
0.637
0.602 0.599
0.630 0.630
0.664 0.654 (I.657
1;q: lc9/18 : lc%PC
(I.585
0.619
(t.630
319
-'°Io
I
I
I
I
I
Y
~-8
oG
> D
g:
I
I
I
l
I
10
20
30
~0
50
mol
%
Cholesterol
Fig. 2. Effect of cholesterol on the relative microviscosity(rl/rl(~)in phosphatidylcholine vesicles. Average values of two to three independent experiments. The deviations between individual values were about 10%. 18: 1c9/18:Icg-PC (O), 16:0/18:1c6-pc (A), 16:0/18: lcg-PC ([~), 16:0/18:lcU-pc (V) at 37°C. 16:0/16:0-sn3-PC (+), 16:0/16:0-rac-PC (.), 16:0/16:0-sn-PC (x) at 47°C.
smaller differences between the three 16:0/18:1-PC species (data not shown). The properties of multilamellar dispersions of the different phospholipids and their mixtures with cholesterol were also probed with spin-labeled stearic acid carrying the reporter group at the C5 position. Order p a r a m e t e r s calculated from the E P R spectra are summarized in Table I. The data indicate that the change in conformational order resulting from incorporation of cholesterol into 16:0/16:0-PC bilayers is insensitive to the chirality of the phospholipid. Spectra from samples with the same cholesterol content were essentially superimposable, in particular with respect to line widths and relative intensities. This implies similar dynamic properties of the probe in the different systems. The same insensitivity to cholesterol holds for the double bond isomers of the 16:0/18: 1-PC species.
Discussion Detailed models for phospholipid-cholesterol interactions envision inter alia specific hydrogen bonding in the vicinity of the polar region of the bilayer [11, 12]. It seems reasonable to assume that any such interaction would be sensitive to the stereochemistry of the phospholipid chiral center. In the few studies that have examined the interaction of sterols with optical
320 antipodes of phosphatidyicholine, no differences could be detected in monolayer compression, nuclear magnetic resonance spectra or differential scanning calorimetry [23, 26-28]. These negative findings have been verified by fluorescence and E P R techniques in the present study. One report claims a differentiating line-broadening effect of cholesterol on the proton nuclear magnetic resonance polymethylene signal of the two enantiomers of 16:0/16:0-PC [29]. This result has however been questioned more recently [27]. Thus techniques probing a broad spectrum of both bulk and molecular membrane properties have failed to provide any evidence for interactions between cholesterol and any element at or near the phospholipid chiral center; therefore they cannot contribute significantly to the condensing effect of cholesterol. This conclusion extends and is also in agreement with a variety of observations showing specifically that the phospholipid ester carbonyl oxygens are not involved in hydrogen bonding [30-36]. It needs to be stressed however, that our results do not rule out chiral recognition between cholesterol and phosphatidylcholine per se, but only that this recognition is not expressed in changes of the membrane physical state. It further follows that chiral recognition is of minor importance not only between different molecules in the bilayer, i.e. between cholesterol and phospholipid but also between the two optical antipodes of phosphatidylcholine. The various physical properties such as microviscosity, phase transition, conformational order and chain dynamics are the same for 16:0/16:0rac-PC as for each individual enantiomer. The condensing effect of cholesterol is generally ascribed to a tight packing of phospholipid acyl chains with the sterol nucleus. According to a more specific proposal, unsaturated acyl chains can participate in attractive van der Waals interactions with both planes of the sterol nucleus, whereas saturated chains interact preferentially with the planar c~-face [12]. The model furthermore suggests that an unsaturated chain interacting with the sterol/3-face is locked as a result of a conformational (t/g) change such that the axial methyl groups at C10 and C13 of the sterol nucleus are enclosed in a 'hydrophobic pocket'. It then follows that a phospholipid with one saturated and one unsaturated chain would pack most closely with cholesterol if the double bond is located in mid position (C18: lC9). This hypothesis is not supported by the present experimental results. In fact, cholesterol exerts the greatest rigidifying effect on fully saturated phosphatidylcholine and the least on phosphatidylcholine containing a double bond in both acyl chains (Fig. 2). Furthermore in the 16:0/18:1-PC series, the position of the double bond was immaterial (Table I) whereas the model [12] would predict a disrupting effect when the double bond is moved from the z19 to either the A 1t and especially the A 6 position. The condensation of phosphatidylcholine membranes as measured here by fluorescence and E P R techniques is substantially tolerant to structural
321
modifications in the phospholipid molecule. This is not surprising since phospholipid acyl chains are sufficiently flexible to compensate for or avoid arrangements unfavorable for cholesterol-phospholipid interactions. The flexibility of the side chains and the possibility of reorienting the entire molecule with respect to the sterol will also allow the system to minimize its free energy by optimizing hydrophobic interactions. On the macroscopic and submacroscopic level, phospholipids in model membranes behave achirally. Achiral behavior was recently found also for phospholipid stimulation of enzymes [37, 38]. However it does not follow that the lipid matrix of the biological membrane is achiral with respect to all of its functions. Thus chiral recognition was recently observed between phospholipids in an electron diffraction study on single microcrystals of 16:0/16:0-PC. Distinct differences were found in the two-dimensional lattice when the optically pure sn-3 isomer and the corresponding racemic compound were compared [39].
Acknowledgement This work was supported by grants-in-aid from the United States Public Health Service and the National Science Foundation.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
L.D. Frye and M. Ededin, J. Cell Sci., 7 (1970) 319. J.S. Wiley and R.A. Cooper, Biochim. Biophys. Acta, 413 (1975) 425. G. Edelman, Science (Washington, DC), 192 (1976) 218. H.J. Heiniger, A.A. Kandutsch and H.W. Chen, Nature (London), 263 (1976) 515. K.A. Ferguson, F.M. David, R.L. Conner, J.R. Landrey and F.B: Mallory, J. Biol. Chem., 250 (1975) 6998. C.E. Freter, R.C. Ladenson and D.F. Silbert, J. Biol. Chem., 254 (1979) 6909. T.M. Buttke, S.D. Jones and K. Bloch, J. Bacteriol., 144 (1980) 124. R.A. Demel, K.R. Bruckdorfer and L.L.M. van Deenen, Biochim. Biophys. Acta, 255 (1972) 31l. C.E. Dahl, J.S. Dahl and K. Bloch, Biochemistry, 19 (1980) 1462. N.P. Franks, J. Mol. Biol., 100 (1976) 345. H. Brockerhofl, Lipids, 9 (1974) 645. C. Huang, Lipids, 12 (1977) 348. H. Brockerhoff, Methods Enzymol., 35 (1975) 315. C.M. Gupta, R. Radhakrishnan and H.G. Khorana, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 4315. Z. Selinger and Y. Lapidot, J. Lipid Res., 7 (1966) 174. E. Baer and J. Maurukas, J. A m . C h e m . Soc., 74 (1952) 158. E. Baer and M. Kates, J. A m . Chem. Soc., 72 (1950) 942. M. Shinitzky and M. Inbar, J. Mol. Biol., 85 (1974) 603.
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W.L. Hubbell and H.M. McConnell, J. Am. Chem. Soc., 93 (19711 314. B.H. Lentz, Y. Barenholz and T.E. Thompson, Biochemistry, 15 (1976) 4521. H.J. Hinz and J.M. Sturtevant, J. Biol. Chem., 247 (19721 6071. E.H. Shimskick and H.M. McConnell, Biochemistry. 12 (19731 2351. P.J, Davis, B.D. Fleming, K.P. Coolbear and K.M.W. Keough, Biochemistry, 2(1 (19811 3633. M. Shinitzky and M. lnbar, Biochim. Biophys. Acta, 433 (19761 133. M. Shinitzky and I. Yuli, Chem. Phys. Lipids, 30 (1982) 261. D. Gosh, R.L. Lyman and J. Tinoco, Chem. Phys. Lipids, 7 (1971) 173. E.M. Arnett and J.M. Gold, J. Am. Chem. Soc., 104 (1982) 636. A. Hermetter and F. Paltauf, Chem. Phys. Lipids, 31 (19821 283. N. Chatterjie and H. Brockerhoff, Biochim. Biophys. Acta, 511 (19781 116. S.F. Bush, H. Levin and l.W. Levin, Chem. Phys. Lipids, 27 (1980) 101. R. Bittman, S. Clejan, M.K. Jain, P.W. Deroo and A.F. Rosenthal, Biochemistry, 20 (19801 2790. A, Hermetter and F. Paltauf, Chem. Phys. hipids, 29 (1981) 225. S. Clejan. R. Bittman, P.W. Deroo, Y.A. lsaacson and A.F. Rosenthal, Biochemistry, 18 (t979) 2118. B. de Kruyff, R.A. Demel, A.J. Slotboom and K.M.W. Keogh, Biochemistry, 20 (1981) 3633. V.V. Chupin, I.A. Vasilenko, G.I. Merkushkin, G.A. Serebrennikova and R.P. Evstigneeva, Bioorg. Khim., 5 (1979) 1515. S. Rainier, M.K..lain, F. Ramirez, P.V. loannu. J.F. Marecek and R. Wagner, Biochim. Biophys. Acta, 558 (1979) 187. Y.A. lsaacson, P.W. Deroo, A.F. Rosenthal, R. Bitterman, J.O. Mclntyrc, H.G. Bock. P. Gazzotti and S. Fleischer, J. Biol. Chem., 254 (1979) 117. A. Joutti, P. Vainio, J.R. Brotherus, F. Paltauf and P.K.J. Kinnunen, Chem. Phys. Lipids. 29 (1981) 235. 1. Sakurai, S. Sakurai, T. Sakurai, T. Seto, A. Ikegami and S. Iwayanagi, Chem. Phys. Lipids, 26 (1980) 41.
313
PHOSPHATIDYLCHOLINE AND CHOLESTEROL INTERACTIONS IN MODEL MEMBRANES
W. G U Y E R and K. B L O C H
James Bryant Conant Laboratories, Department of Chqmistry, Harvard University, Cambridge, MA 02138 (U.S.A.) Received D e c e m b e r 8th, 1982
accepted April 30th, 1983
Various phosphatidylcholines differing either in the stereochemistry around their chiral center or in the position of a cis double bond along the acyl chains were synthesized in order to study critical contact regions in the phospholipid molecule with adjacent cholesterol in model m e m b r a n e s . Microviscosities calculated from fluorescence depolarization of diphenylhexatriene and chain order from spin label studies were m e a s u r e d to monitor physical m e m b r a n e properties. T h e enhancing effect of cholesterol on the microviscosity of m e m b r a n e s containing phosphatidylcholines with comparable acyl chain length was largest when the two acyl chains were saturated and smallest when both were unsaturated. M e m b r a n e s prepared from phosphatidylcholines having a single cis double bond at different positions along the sn-2 acyl chain showed roughly the same changes of microviscosity or chain order upon incorporation of cholesterol. No discrimination was evident in the interaction between cholesterol and enantiomeric phosphatidylcholines or between the enantiomeric phosphatidylcholine molecules themselves. We conclude that the rigidifying effect of cholesterol in m e m b r a n e s does not depend on specific sites of interaction and that with respect to physical m e m b r a n e properties phosphatidylcholine behaves as an achiral molecule.
Keywords: lipid-lipid interaction; phosphatidylcholine; cholesterol; structural specificity; chiral recognition.
Introduction Sterols and phospholipids are the major lipids of cellular membranes, and it appears that for cell growth and function, a balanced ratio of the two components has to be maintained. At the physiological or biochemical level the function of sterols in membranes is not well understood. However, experimentally induced changes in the sterol concentration have been shown to alter the physical state of the membrane and in turn modulate a broad range of cellular processes [1-4]. Such changes of physical state are often accompanied by adjustments in the structure of the phospholipid molecule [5-7]. Various physiological responses are believed to be related in some manner to membrane fluidity which in turn is controlled by the dynamic 0009-3084/83/$03.00 O 1983 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
314 interactions between the various lipid components in the hydrophilic polar zone and the hydrophobic core. The structure of cholesterol appears to be especially well designed for modulating the physical state of phospholipid bilayers [8, 9]. Evidence based on a variety of physical techniques has been interpreted as showing that cholesterol entering the membrane aligns in parallel with the extended acyl chains of the phospholipids, the sterol hydroxyl group pointing toward the water-bilayer interface [10]. Since both sterol and phospholipid are chiral but only one enantiomer of each is commonly found in nature, stereochemical factors could conceivably be of importance for the strength of interaction between the two lipid components. Some current, more explicit membrane models specify distinct regions of interactions, namely (a) hydrogen bonding between the sterol hydroxyl function and some polar element of the phospholipid head group, e.g. the ester carbonyl [1 1, 12] and (b) hydrophobic interactions in the core of the bilayer which are postulated to be sensitive to the number and location of double bonds in the phospholipid acyl chains [12]. In the present study we describe rigidifying effects of cholesterol in membrane bilayers prepared from various synthetic positional isomers and enantiomers of phosphatidylcholine. These phosphatidylcholines were designed to detect perturbations in the interactions involving either the polar or the hydrophobic contact regions of cholesterol.
Materials and methods
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (16 : 0/16:0-sn3-PC), t,2dipalmitoyl-rac-glycero-3-phosphocholine (16:0/16:0-rac-PC) and 1,2dioleoyl-sn-glycero-3-phosphocholine (18: lc9/18: lc9-pc) were purchased from Sigma Chemical Co. 2,3-Dipalmitoyl-sn-glycero- 1-phosphocholine (16 : 0/16 : O-sn 1-PC) was prepared from 16 :0/16 :0-rac-PC by enzymatic hydrolysis of the sn-3 enantiomer with phospholipase A2 (lyophilized Crotalus adamanteus venom from Sigma Chemical Co.) [13], leaving the sn-1 enantiomer intact. The resulting crude lipid mixture was taken to dryness by rotary evaporation and the sn-1 enantiomer purified by chromatography on silica gel (Unisil Clarkson Chemical Co) (100g/g lipid) with chloroform/methanol as solvent. The phospholipid fraction was passed through a column of Rexyn 1-300 mixed bed ion exchanger (Fisher Scientific Co.) (50 ml/mmol phosphatidylcholine) with chloroform/methanol/water (4 : 5 : 1, by vol.) as the solvent system. This step removed any possible contamination with Ca 2+ remaining from the phospholipase A2 treatment. The phospholipid was rechromatographed on a silica gel column as above and finally crystallized from acetone/chloroform.
315
1-Palmitoyl-2-petroselinoyl-sn-glycero-3-phosphocholine (16:0/18 : lc 6PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0/18 : lcg-PC) and 1-palmitoyl-2-vaccenoyl-sn-glycero-3-phosphocholine (16:0/18 : lc11-PC) were synthesized by enzymatic removal of the sn-2 chain from 16:0/16:0sn3-PC with phospholipase A2 [13] and reacylation with the appropriate fatty acid anhydrides under conditions that minimize acyl chain migration [14[. Petroselinic, oleic and vaccenic acids were from Sigma Chemical Co. (nominal purity > 9 8 % ) and the various anhydrides were prepared [15]. All phospholipids were purified by chromatography on Rexyn 1-300 and silica gel and crystallized as outlined above. In order to prevent air oxidation, all steps were carried out under nitrogen. A trace of hydroquinone was added to the solvents throughout synthesis and purification, except for the final crystallization step. The pure phospholipids were stored under argon at -2{}°C. The chemical purity of the phospholipids was checked by TLC. Samples of {}.4 mg of the phospholipids were applied to TLC plates (Si250, J.T. Baker Chemical Co.) and developed with the solvent systems CHCI3/CH3OH/ NH4OHaq (65:28:4, by vol.) and CHCI3/CH3OH/CH3COOH/H20 (50:25:7:3, by vol.) respectively over a distance of 18 cm. Only single spots could be detected upon exposure to iodine or after charring with sulfuric acid. Greater than 98% of the applied sample was recovered from the phospholipid zone as determined by phosphate analysis. The optical purity of 16:0/16:0-PC was verified by measuring rotations with a Perkin Elmer 141 polarimeter. 16:0/16:0-sn3-PC, [a]~ = + 6 . 3 __~_0 . 2 o , 16:0/16:(}-snl-PC [c~]~ = -6.4+_0.2°; (c = 1.6, CHCI3/CH3OH, 1:1). 16:0/16:{}-rac-PC exhibited no optical rotation. Previously reported values for 16:(}/16:0-sn3-PC range from [a]~=+7.{} ° ( c = 5 . 6 , CHC13) [16] to [a]~ = +6.6 ° (c = 8, CHCI3/CH3OH, 1" 1) [t7]. Cholesterol (Aldrich Chemical Co.) was recrystallized twice from 95% ethanol and acetone and dried in vacuo. The fluorescent probe, 1,6diphenyl-l,3,5-hexatriene (DPH) and the spin-labeled fatty acid 5-doxylstearic acid were purchased from Aldrich Chemical Co. and Syva Associates, respectively, and used as supplied. Preparation of liposomes The required amounts of lipids were dissolved in CHCI3/CH3OH (2: 1, by vol.) and the mixture was dried under a stream of nitrogen. Residual solvent was removed in vacuo in a desiccator containing K O H pellets and P20~. For microviscosity measurements of multilamellar dispersions, the aqueous medium (50 mM NaCI, 10 mM sodium phosphate buffer (pH 7.2), 1 mM E D T A ) was supplemented with 10% sucrose, added to the lipid film and the lipids dispersed by shaking on a Vortex mixer for 5 rain above the transition
316 t e m p e r a t u r e (To) of the phospholipid. These dispersions were equilibrated for at least 3 h at the t e m p e r a t u r e chosen for subsequent experiments. Unilamellar vesicles were prepared by sonication with a Heat Systems sonifier Model W-350 (at 20 kHz, 70 W output power, 50% duty cycle) for 15 rain in a conical glass tube. The tip of the soniprobe was immersed to about half of the dispersion height. During sonication, the tube was kept in a water bath above T,. and a stream of H20-saturated nitrogen was passed over the dispersion. No degradation of the lipids could be observed by TLC. Titanium from the soniprobe was removed from the sample by centrifugation for 10 min at 2000 × g and the clear supernatant was kept above T,. and used the same day. Vesicle yield was typically better than 85% as determined by sizing on a column of Bio-Gel A-50m (Bio-Rad Laboratories). Microviscosity measurements Microviscosities were determined in an Elscint microviscosimeter Model MV-1A, with D P H as the fluorescent probe. To 2.5 ml of the lipid suspension (1.25 p~mol total lipid) was added 1 ~1 D P H (1 mM in tetrahydrofuran) with vigorous shaking. This mixture was incubated at 37°C (47°C for 16:0/16 : 0-PC) for 60 min. Fluorescence depolarization was measured at the temperatures indicated under results, and microviscosities calculated according to Shinitzky and Inbar [18]. Electron paramagnetic resonance E P R spectra were recorded at the indicated temperatures on a Varian E-line 9.5 G H z spectrometer equipped with a Varian t e m p e r a t u r e controller. The microwave power was held constant at 20 m W and the modulation amplitude at 0.8 G. Aliquots of 50 ~1 multilamellar lipid dispersions prepared from 2 ~mol total lipid were placed in a 1 m m glass capillary and E P R spectra were recorded. Spin-labeled fatty acids were introduced into the liposomes at a molar ratio of 1:200. 2T~1and 27"1 were derived from the spectra by measuring the splittings between the outer and the inner hyperfine peaks and the order p a r a m e t e r (S) was calculated from these values according to Hubbell and McConneli [19].
Results
Microviscosity values derived from the fluorescence depolarization of D P H provide valid, comparative measures of m e m b r a n e 'fluidity' as a function of m e m b r a n e composition. Figures 1A and B illustrate the effect of
317 TEMPERATURE 50 20
]
40 i
I
30 i
I
20 q
I
i
]o
5
I
T
(°C) ~
a.
30
20
lO
F
I
]
5
t
b.
10 ~
50
>I,- 20 0 10 u') 05 31
32
]3
34
I
I
1
I
I
I
I
35
36
32
33
3.4
3.5
36
3
(°K-l)
1/T.10
Fig. 1. Effect of temperature on the microviscosity(-q) of multilamellar dispersions of phosphatidylcholines (open symbols) and their 1 : 1 mixtures with cholesterol (closed symbols). Averaged data of two to four independent experiments. (a) 18: lc9/18: lc9-pc (©, O); 16:0/16:0-sn3-PC (A, &); 16:0/16:0-rac-PC ([], i ) ; 16:0/16:0-snl-PC (V, V). (b) 16:0/18:1c6-pc (A, &); 16:0/18: lc9-pc ([2, I ) ; 16:0/18: lclLPC (~', V).
t e m p e r a t u r e on the microviscosity of multilamellar lipid dispersions prepared from the synthetic phosphatidylcholines and their 1:1 mixtures with cholesterol. The data are presented as Arrhenius plots. In the t e m p e r a t u r e range studied all phosphatidylcholine-cholesterol mixtures and three of the unsaturated pure phosphatidylcholines show the expected linear relation. In both phospholipids alone and in their 1:1 mixtures with cholesterol, the presence of an unsaturated phospholipid acyl chain causes a decrease of microviscosity. A m o n g the 'mixed' phospholipids, 16:0/18: lc9-pc containing the double bond in the central position along the acyl chain yields the most fluid bilayer. In the absence of cholesterol abrupt changes in slope are observed for the three optically different species of 16:0/16:0-PC (Fig. 1A) and for 16:0/18: lc6-pc (Fig. 1B) attributable to the gel to liquid-crystalline phase transition [20]. The important result is that the curves for the two enantiomers of 16:0/16:0-PC and their racemic mixture are indistinguishable from each other and that all three show a transition t e m p e r a t u r e (midpoint of the change) of Tc = 40.8°C (range 39.3-42.4°C) (Fig. 1A) in good agreement with published values for 1 6 : 0 / 1 6 : 0 - s n 3 - P C [20-22]. For the 16:0/18: Ic6-pc, not previously reported, T~ was 19.4°C (range 18.521.3°C) (Fig. 1B). This value is high c o m p a r e d to those for 16:0/18: lc9-pc and 16:0/18 : lclS-pc, which show a phase transition at a t e m p e r a t u r e lower than the range chosen. The published value for 16:0/18: lc9-pc is Tc < 5 ° C
318
[231. The 'smoothing' effect of cholesterol on the phase transition evident in Figs. 1A and 1B has been observed repeatedly with various methods. Qualitatively similar curves were obtained with unilamellar vesicles (data not shown). Incorporation of increasing amounts of cholesterol into vesicles progressively raises m e m b r a n e microviscosities above Tc [24]. Figure 2 shows the microviscosity increase caused by incremental quantities of cholesterol in unilamellar vesicles prepared from the various phosphatidylcholines. Since the intrinsic microviscosities of the pure phospholipids were different, relative microviscosities were calculated. This presentation of the data seems justified by the finding that microviscosities measured in phosphatidylcholine-cholesterol bilayers by the D P H method obey the Batschinsky relation, which inversely links microviscosity to the free volume in the lipid [25]. Cholesterol raises the microviscosity of 16:0/16:0-PC markedly producing 11 times the value shown by the pure phospholipid at a content of 5[) m o l % . H o w e v e r the responses of the three optically different forms of 16:0/16:0-PC were identical as shown by the three superimposable curves in Fig. 2. The effect of cholesterol on the unsaturated phosphatidylcholines was in general less marked, the di-unsaturated 18:1c9/18 : lcg-PC being least affected. As for the phosphatidylcholines with one unsaturated acyt chain in the sn-2 position, a shift of the central double bond in either direction along the chain only slightly altered the susceptibility to cholesterol. Qualitatively similar results were obtained with multilamellar lipid dispersions with even
TABLE 1 O R D E R P A R A M E T E R S (S) O F P H O S P H O L I P I D A N D P H O S P H O L I P I D CHOLESTEROL MULTILAMELLAR DISPERSIONS The estimated error of $ is +(I.()1. Temperature, 47°(7 for 16:0/16:0-PC, 37°C for all other phospholipids. Cholest erol/phospholipid
16:0/16:0-sn3-PC 16:0/16:0-rac-PC 16 : 0/16 : 0-sn 1-PC
0
1:4
1:2
[).571 0.569 0.565
0.625 0.619 (I.625
0.665 (I.658 [I.665
16 : 0 / 1 8 : l c ~ - P C 16:0/18: Icg-PC 16:0/18: IcLLPC
0.596
0.637
0.602 0.599
0.630 0.630
0.664 0.654 (I.657
1;q: lc9/18 : lc%PC
(I.585
0.619
(t.630
319
-'°Io
I
I
I
I
I
Y
~-8
oG
> D
g:
I
I
I
l
I
10
20
30
~0
50
mol
%
Cholesterol
Fig. 2. Effect of cholesterol on the relative microviscosity(rl/rl(~)in phosphatidylcholine vesicles. Average values of two to three independent experiments. The deviations between individual values were about 10%. 18: 1c9/18:Icg-PC (O), 16:0/18:1c6-pc (A), 16:0/18: lcg-PC ([~), 16:0/18:lcU-pc (V) at 37°C. 16:0/16:0-sn3-PC (+), 16:0/16:0-rac-PC (.), 16:0/16:0-sn-PC (x) at 47°C.
smaller differences between the three 16:0/18:1-PC species (data not shown). The properties of multilamellar dispersions of the different phospholipids and their mixtures with cholesterol were also probed with spin-labeled stearic acid carrying the reporter group at the C5 position. Order p a r a m e t e r s calculated from the E P R spectra are summarized in Table I. The data indicate that the change in conformational order resulting from incorporation of cholesterol into 16:0/16:0-PC bilayers is insensitive to the chirality of the phospholipid. Spectra from samples with the same cholesterol content were essentially superimposable, in particular with respect to line widths and relative intensities. This implies similar dynamic properties of the probe in the different systems. The same insensitivity to cholesterol holds for the double bond isomers of the 16:0/18: 1-PC species.
Discussion Detailed models for phospholipid-cholesterol interactions envision inter alia specific hydrogen bonding in the vicinity of the polar region of the bilayer [11, 12]. It seems reasonable to assume that any such interaction would be sensitive to the stereochemistry of the phospholipid chiral center. In the few studies that have examined the interaction of sterols with optical
320 antipodes of phosphatidyicholine, no differences could be detected in monolayer compression, nuclear magnetic resonance spectra or differential scanning calorimetry [23, 26-28]. These negative findings have been verified by fluorescence and E P R techniques in the present study. One report claims a differentiating line-broadening effect of cholesterol on the proton nuclear magnetic resonance polymethylene signal of the two enantiomers of 16:0/16:0-PC [29]. This result has however been questioned more recently [27]. Thus techniques probing a broad spectrum of both bulk and molecular membrane properties have failed to provide any evidence for interactions between cholesterol and any element at or near the phospholipid chiral center; therefore they cannot contribute significantly to the condensing effect of cholesterol. This conclusion extends and is also in agreement with a variety of observations showing specifically that the phospholipid ester carbonyl oxygens are not involved in hydrogen bonding [30-36]. It needs to be stressed however, that our results do not rule out chiral recognition between cholesterol and phosphatidylcholine per se, but only that this recognition is not expressed in changes of the membrane physical state. It further follows that chiral recognition is of minor importance not only between different molecules in the bilayer, i.e. between cholesterol and phospholipid but also between the two optical antipodes of phosphatidylcholine. The various physical properties such as microviscosity, phase transition, conformational order and chain dynamics are the same for 16:0/16:0rac-PC as for each individual enantiomer. The condensing effect of cholesterol is generally ascribed to a tight packing of phospholipid acyl chains with the sterol nucleus. According to a more specific proposal, unsaturated acyl chains can participate in attractive van der Waals interactions with both planes of the sterol nucleus, whereas saturated chains interact preferentially with the planar c~-face [12]. The model furthermore suggests that an unsaturated chain interacting with the sterol/3-face is locked as a result of a conformational (t/g) change such that the axial methyl groups at C10 and C13 of the sterol nucleus are enclosed in a 'hydrophobic pocket'. It then follows that a phospholipid with one saturated and one unsaturated chain would pack most closely with cholesterol if the double bond is located in mid position (C18: lC9). This hypothesis is not supported by the present experimental results. In fact, cholesterol exerts the greatest rigidifying effect on fully saturated phosphatidylcholine and the least on phosphatidylcholine containing a double bond in both acyl chains (Fig. 2). Furthermore in the 16:0/18:1-PC series, the position of the double bond was immaterial (Table I) whereas the model [12] would predict a disrupting effect when the double bond is moved from the z19 to either the A 1t and especially the A 6 position. The condensation of phosphatidylcholine membranes as measured here by fluorescence and E P R techniques is substantially tolerant to structural
321
modifications in the phospholipid molecule. This is not surprising since phospholipid acyl chains are sufficiently flexible to compensate for or avoid arrangements unfavorable for cholesterol-phospholipid interactions. The flexibility of the side chains and the possibility of reorienting the entire molecule with respect to the sterol will also allow the system to minimize its free energy by optimizing hydrophobic interactions. On the macroscopic and submacroscopic level, phospholipids in model membranes behave achirally. Achiral behavior was recently found also for phospholipid stimulation of enzymes [37, 38]. However it does not follow that the lipid matrix of the biological membrane is achiral with respect to all of its functions. Thus chiral recognition was recently observed between phospholipids in an electron diffraction study on single microcrystals of 16:0/16:0-PC. Distinct differences were found in the two-dimensional lattice when the optically pure sn-3 isomer and the corresponding racemic compound were compared [39].
Acknowledgement This work was supported by grants-in-aid from the United States Public Health Service and the National Science Foundation.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
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