Phase transitions and fluidity characteristics of lipids and cell membranes.

Quarterly Reviews of Biophysics 8, 2 (1975), pp. 185-235 Printed in Great Britain

Phase transitions and fluidity characteristics of lipids and cell membranes D. CHAPMAN Chemistry Department, Sheffield University, Sheffield S3 7HF

I. INTRODUCTION II. T H E R M O T R O P I C PHASE TRANSITIONS OF L I P I D S

186 187

A. Anhydrous lipids 187 The significance of the thermotropic phase transition of anhydrous lipids 194 B. Hydrated lipids 195 1,2-diacyl-L-phosphatidylcholines (lecithins) 196 Egg-yolk lecithin 199 Other lipid-water systems 204 Hydrocarbon chain organization 205 C. Correlation with monolayer properties 207 D. Phase separation 209 E. Cholesterol effects 212 F. Metal ion effects 214 G. Protein and polypeptide effects 215 H. Drug interactions 217 I. Permeability characteristics 218 J. Theoretical treatments 218 III.

THERMOTROPIC PHASE TRANSITIONS OF CELL

MEMBRANES

A. B. C. D.

Myelin membranes 219 Erythrocyte membranes 220 Acholeplasma laidlawii membranes 220 Escherichia coli membranes 221

219

l86

D. CHAPMAN

E. Yeast membranes 222 F. Other membranes 223 IV. THE SIGNIFICANCE OF LIPID PHASE TRANSITIONS FOR BIOLOGICAL SYSTEMS

224

A. Fluidity 224 B. Trigger mechanisms 225 C. Cryobiology 226 V. ACKNOWLEDGEMENTS VI. REFERENCES

226 226

I. INTRODUCTION

The concept that the liquid crystalline or mesomorphic condition was of importance to biological systems is a relatively old idea. Thus Bernal (1933) when discussing the different types of arrangements of molecules in liquid crystals commented 'Such structures belong to the liquid crystal as a unit and not to its molecules which may be replaced by others without destroying them and they persist in spite of the complete fluidity of the substance. These are just the properties to be required for a degree of organization between that of the continuous substance, liquid or crystalline solid and even the simplest living cell.' Stewart (1961) some thirty years later also stated that ' It is this property - the combination offlowand lability with a preferred and relatively stable molecular orientation - that makes the mesomorphic (i.e. liquid crystal) phase uniquely appropriate to the structure of protoplasm and living tissue.' The lipid components of cell membranes have this ability to form a mesomorphic condition and to form a periodically ordered long-range organization with a somewhat disordered short-range organization. These mesomorphic properties are particularly important for cell membrane organization and function and underlie to some extent our present-day concepts of fluidity and phase transition properties now commonly used to describe cell membrane structures. It is interesting to reflect that Schmitt, Bear & Clark as long ago as 1935 interpreted their X-ray diffraction studies of myelin in terms of oriented fluid crystals with the lipids organized in a mixed crystal fashion. A whole range of physical techniques have now been applied to the

Phase transitions and fluidity characteristics

187

study of the thermotropic phase transition of anhydrous and hydrated lipid systems. These include X-ray methods, calorimetry, infrared (i.r.) and Raman spectroscopy, nuclear magnetic resonance (n.m.r.) spectroscopy, light scattering, and dilatometry. Various probe molecules have now been included in lipid systems including fluorescent probes, spin label probes, and also triplet probes. Each of the techniques has its own advantages and limitations which always need prior careful consideration, for the particular problem involved. In this review paper we intend to examine what is physically involved when phase transitions occur with lipid systems, and the way in which these phase changes are related to the fluidity and phase separation characteristics of membrane systems. We shall also examine the trigger mechanisms which can change phase transition temperatures and affect membrane and cell functions. Finally we shall point to other implications which have not yet been explored which these lipid phase transitions may have for biological systems. II. T H E R M O T R O P I C PHASE TRANSITIONS OF L I P I D S

A. Anhydrous lipids

Thermotropic transitions (i.e. phase/changes caused by the effect of heat) of lipid type molecules have been studied for many years. The complex mesomorphism of soap systems in particular received much early attention (Lawrence, 1938; Void, 1941). With anhydrous sodium palmitate, five phase transitions were observed between the crystalline state and the isotropic melt (Void, Macomber & Void, 1941). The various regions were referred to as curd to waxy, waxy to subneat, subneat to neat, and neat to isotropic. Nordsieck, Rosevear & Ferguson (1948) examined these systems using X-ray methods and showed that sharp, short spacings occur up to n o °C and above this temperature that the short spacings were diffuse. They considered that their data showed that there was order in the long spacing direction and liquid or amorphous order laterally. The 4-8 A halo was interpreted to indicate hydrocarbon chains in a loose hexagonal packing arrangement. Palmer & Schmitt (1941) examined nerve lipids using X-ray diffraction in the dry and wet conditions. In the dry state the lipids gave a short spacing at 4-2 A but on wetting a diffuse spacing at 4-6 A was produced which they associated with the hydrocarbon phase being

l88

D. CHAPMAN TABLE I.

Summarized melting-point data of phospholipids Melting point °C

i ,2-diacylphosphatidylcholine

230

1,2-diacylphosphatidylethanolamine i ,3-diacylphosphatidylethanolamine i ,2-diacylphosphatidylserine

196

i ,2-diacylphosphatidylinositol

136

1,2-diacylphosphatidyIglycerol

67

1,2-diacylphosphatidic acid

7i

1,3-diacylphosphatidic acid

70

bis phosphatidic acid

70

198 160

Sphingomyelins

210

Lysophosphatidylcholine

240

Lysophosphatidylethanolamine

205

Comments Melting point practically independent of chain length Melting point practically independent of chain length Melting point practically independent of chain length Shorter chain lengths; lower melting points Insufficient data to determine melting point chain length dependency Insufficient data to determine melting point chain length dependency Shorter chain lengths; lower melting points Shorter chain lengths; lower melting points Insufficient data to determine melting point chain length dependency Insufficient data to determine melting point chain length dependency Insufficient data to determine melting point chain length dependency Insufficient data to determine melting point chain length dependency

essentially liquid in character. Later, Finean (1953) and Finean & Millington (1955) examined the effects of heat upon a series of pure lipid systems using X-ray methods. They observed changes in the X-ray long spacings and short spacings which they interpreted in terms of the production of various forms having different tilting of the long axes of these molecules. They ruled out other configurations of the hydrocarbon chains than the planar zig-zag arrangement to explain their data. The detailed molecular nature of the major thermotropic phase transition of these long-chain amphiphilic molecules was delineated by i.r. spectroscopic studies (Chapman, 1958). This showed that on heating anhydrous sodium palmitate above 120 °C the i.r. spectra changed dramatically from that typical of a crystal to that typical of a liquid consistent with the occurrence of rotational isomers produced by methylene group rotation about the carbon-carbon bonds. Raising the temperature caused a further decrease in the intensity of a band at 719 cm"1 (associated with the CH2 rocking mode) in the spectrum show-

Phase transitions and fluidity characteristics 189

ing that a further decrease in the all-transconfiguration occurs at higher temperatures. Thus the liquid-like character or fluidity of the hydrocarbon chains above a certain transition temperature was established and related to the liquid crystalline phases of these molecules. In further studies of pure phospholipids using a range of physical techniques - i.r. spectroscopy, X-ray, microscope methods, and n.m.r. spectroscopy - a series of phosphatidylethanolamines and phosphatidylcholines (lecithins) showed a similar mesomorphic behaviour to those of the simple soap systems (Chapman, Byrne & Shipley 1966; Chapman, Williams & Ladbrooke, 1967). When we examine the melting points of the different classes of phospholipids, we see a large variation (Table 1). On the one hand the phosphatidylglycerols, the phosphatidic acids and the bis phosphatidic acids have melting points which are chain-length dependent, and which do not exceed about 70 °C. For these phospholipids the shorter or more unsaturated the hydrocarbon chains, the lower the melting point. On the other hand, the phosphatidylethanolamines, phosphatidylcholines and sphingomyelins have melting points of about 200, 230, and 210 °C, respectively, and these do not appear to be significantly affected by the type of fatty acyl residues present. Intermediate between these extremes are the phosphatidylserines, the melting points of which appear to be chain-length dependent, and the phosphatidylinositols, about which sufficient data are not available to know whether their melting points are chain-length dependent. The phosphatidic acids, which have low melting points, do not exhibit mesomorphic behaviour. Their melting points are of the same order as those of the corresponding chain length fatty acids, and from this we may infer that the forces of interaction between the molecules in the crystal are principally due to hydrogen bonds, and are consequently weak. The phospholipids with high melting points, such as the phosphatidylcholines, are more akin to the soaps (fatty acid salts) in their melting behaviour. The soaps are linked together by an ionic network which holds the structure together until high temperatures (300 °C) are reached. It therefore seems likely that an ionic network exists in the crystal structure of these phospholipids. An ionic network would lead us to expect that the stronger the base the higher the melting point, and this is indeed found (see Table 1) in order of decreasing melting point. An examination of a given class of these high melting point phospho-

190

D. CHAPMAN

lipids reveals that the melting points are mainly independent of the fatty acid residues present. For example, i-stearoyl-2-oleoyl- and 1,2-distearoyl-L-phosphatidylcholine both have melting points of 230231 °C. The melting points of these phospholipids are not markedly affected by the number of acyl chains present. The melting points of the lyso-derivatives are slightly higher than those of the corresponding diacyl phospholipids. It is thus quite clear that the polar head group of these phospholipids is the major factor controlling their melting points (Williams & Chapman, 1970). In addition to the capillary melting point, phase changes occur with phospholipids at lower temperatures; e.g. when a pure phospholipid, dimyristoylphosphatidylethanolamine, containing two fully saturated chains, is heated from room temperature to the capillary melting point, a number of thermotropic phase changes occur. This was first shown by i.r. spectroscopic techniques (Byrne & Chapman, 1964), then by thermal analysis (Chapman & Collin, 1965), and has now been studied by a variety of physical techniques (Chapman et al. 1966). Optical studies show that dimyristoylphosphatidylethanolamine at room temperature is birefringent under crossed polars. On heating, three processes occur: first, some loss of birefringence at the first transition temperature ~ 120 °C, then a small increase at 135 °C, and a pronounced overall loss of birefringence near the capillary melting point of 200 °C. Above 120 °C, pressure on a coverglass with a needle causes the material to flow. When the temperature of the phospholipid reaches ~ 120 °C the i.r. absorption spectrum undergoes a remarkable change. Above this temperature the spectrum loses all the fine structure and detail which was present at lower temperatures, and the spectrum becomes similar to that obtained with a phospholipid dissolved in a solvent such as chloroform. The i.r. spectra of this phospholipid at different temperatures are shown in Fig. 1. Differential thermal analysis (d.t.a.) shows that a marked endothermic transition (absorption of heat) occurs at this transition temperature. An additional heat change occurs at 135 °C and only a small heat change is involved near the capillary melting point of the lipid (see Fig. 2). This behaviour is similar to that which occurs with liquid crystals, such as />-azoxyanisole or cholesteryl acetate which form nematic and cholesteric liquid crystalline phases. The X-ray long spacings show a dramatic reduction to some twothirds of their original value at the first transition temperature with a

Phase transitions and fluidity characteristics 191 2500 2700 2100 3Oo'o 2000 1250 4000 00 | | I || 1600 HOP 900 70 700 T III II 1 1 I I I I I 120 °C

1

1

4

1

1

1

1 1

6 8 10 Wavelength (//m) wavcicugiu \fiiu)

12

14

Fig. i. The infra-red spectrum of i,2-dilauroyl-DL-phosphatidylethanolamine at different temperatures (Chapman et al. 1966).

o

It

rv

200

100 Temperature (°C)

Fig. 2. Differential thermal analysis heating curve of 1,2-dimyristoylphosphatidylethanolamine (Chapman & Collins, 1965).

192

D. CHAPMAN

further small reduction at the second transition temperature. The X-ray short spacings change at the first transition point from sharp diffraction lines to a diffuse spacing at ~ 4-6 A. Nuclear (proton) magnetic resonance (p.m.r.) studies of these phospholipids show a gradual reduction in line width from about 15 G at liquid-nitrogen temperature until, at the first transition temperature, there is a sudden reduction in the line width (to ~ 0-09 G). This shows that molecular motion increases gradually as the temperature increases until, at the transition temperature, a considerable increase in the molecular motion takes place (Chapman & Salsbury, 1966). The main conclusions from these various studies are that (a) even with the fully saturated phospholipid at room temperature, some molecular motion occurs in the solid. This is evident from the p.m.r. spectra and from the i.r. spectra taken at liquid-nitrogen and at room temperatures. (Note the difference between the i.r. spectra at — 186 °C and room temperature shown in Fig. 1.) (b) When the phospholipid is heated to a higher temperature, it reaches a transition point, a marked endothermic change occurs, and the hydrocarbon chains in the lipid 'melt' and exhibit a very high degree of molecular motion. This is evident both in the appearance of the i.r. spectrum and also in the narrow n.m.r. line width. On the other hand, the broad diffuse appearance of the i.r. spectrum is consistent with the chains flexing and twisting and with a 'break-up' of the all-planar trans configuration of the chains. When phospholipids contain shorter chain lengths, or unsaturated bonds, those marked endothermic phase transitions occur at lower temperatures. The temperature at which these transitions occur parallels the behaviour of the melting point of the related fatty acids. The transition temperatures are high for the fully saturated long chain phospholipids. They are lower when there is a trans double bond present in one of the chains and lower still when there is a cis double bond present. This variation of transition temperature also confirms that this phase transition is associated with a ' melting' of the hydrocarbon chains of the phospholipid, while this in turn is a reflexion of the dispersion forces between the chains. Only one main' melting of the chains' occurs even when there are two different types of chain present in the phospholipid molecule. The transition temperatures for different phospholipid classes vary even though they contain exactly the same fatty acid residues. While natural


100

« 50

1

1

I

1

I

10 12 14 16 18 20 22 24 26 Hydrocarbon chain length

I

1

28 30

Fig. 3. Transition temperatures of various diacylphosphatidylethanolamines and diacylphosphatidylcholines, both in the anhydrous condition and in the presence of water. The melting points of n-paraffins of the same hydrocarbon chain length are also included for comparison. + , anhydrous 1,2-diacyl-DLphosphatidylethanolamines; • , i ,2,-diacyl-DL-phosphatidylethanolamines in water; A, anhydrous 1,2-diacyl-L-phosphatidylcholines; O, i,2-diacylL-phosphatidylcholine monohydrates (al form); x , 1,2-diacyl-L-phosphatidylcholines in water; • , melting points of normal paraffins (Chapman et al. 1967).

phospholipids from erythrocyte or mitochondrial membranes contain large amounts of unsaturated cis double bonds and, therefore, in the dry condition have endothermic transition temperatures either near or below room temperatures, the highly saturated derivatives exhibit transition temperatures much higher than room temperature (Chapman et al. 1966). The lecithins or phosphatidylcholines have also been extensively studied (Chapman et al. 1967). Series of d.t.a. heating curves for the ax form of the 1,2-diacyl-L-phosphatidylcholine monohydrates have been obtained. In each case a pronounced endothermic transition is observed, corresponding to the melting of the hydrocarbon chains of the lecithins. The temperature of transition is chain-length dependent, as with the phosphatidylethanolamines. Egg yolk lecithin, which gives a much broader endotherm than do the phosphatidylcholines of a single discrete chain length, does not behave as a homogeneous phase at this transition, but behaves as a mixture of chain lengths. Small (1967) 13

QRB 8

194

D- CHAPMAN O

CH 3 (CH 2 ) n _ 2 -C-O-CH 2 CH 3 (CH 2 ) B . 2 -C-O-CH 70

O

O

(+)

CH r O-P-O-CH r CH 2 -N(CH 3 )3

o("

60 0

204

CHAPMAN

S 2

10 -

-30 -10

30

50

70

90

110

130

150

170

Temperature (°C) Fig. 9. The measured DMR splittings and quadrupole coupling constants for various values of n as a function of temperature through the phase transition for dipalmitoylphosphatidylcholine-n DaO (Salsbury et al. 1972). A, 2-4 DjO/mole DPL; O, 5-3 DaO/mole DPL; Q . 173 D2O/mole DPL; x , ai-o, DaO/mole DPL.

because of the high spin label concentrations customarily employed, both dipole-dipole and electron exchange interactions contribute to line broadening. At temperatures below Tc the former predominates and estimation of diffusion rates becomes exceedingly difficult. Triplet probes have been used (Naqvi et al. 1974) to estimate the change in diffusion coefficient of these triplet labelled lipids above and below the transition temperature. The values obtained with sonicated vesicles were i-6 x io~8 cm2 sec"1 at 50 °C and 2-3 x io~7 cm2 sec"1 below the transition temperature of dipalmitoyl lecithin. Other lipid-water systems. A variety of other lipid-water systems


have been examined, notably by Luzzati and co-workers (see ReissHusson). These include egg-yolk phosphatidylethanolamine, sphingomyelin, egg-yolk lysolecithin, a human brain extract (Luzzati & Husson, 1962), a mitochondrial lipid extract (Gulik-Krzywicki et al. 1969), and an erythrocyte lipid extract (Rand & Luzzati, 1968). Luzzati (1968) in particular has pointed out the various other lipid-water forms that can occur with these systems, e.g. hexagonal as well as lamellar phases. The mitochondrial lipid system (Gulik-Krzywicki et al. 1969) is of interest in that with these lipids it was shown that the thermotropic phase transition is a gradual one. It was considered that domains of crystalline chains grow within the bilayers as the temperature is lowered so that part of each chain is 'fluid' and part is crystalline. Levine (1973) has commented on this interpretation. Other lipid-water systems have also been examined (see Shipley, 1973)Hydrocarbon chain organization. The detailed organization of the hydrocarbon chains of lipid mesophases both in the anhydrous and aqueous systems have been variously described as liquid-like and chaotic (Luzzati, 1968), extended and ordered (Vandenheuvel, 1966), significantly ordered (Levine & Wilkins, 1971), and partly ordered with some rotational isomerism (Phillips et al. 1969). The interpretation of the X-ray data associated with the short spacings is not unequivocal (see Levine, 1973). The diffuse diffraction band at 4-6 A from randomly oriented bilayers is similar to that obtained from liquid paraffins. This diffraction is too complex to be analysed in terms of a detailed model for the organization of the hydrocarbon chains, and it is yet to be shown that it corresponds to a unique mode of packing of the chains. Warren (1933) has discussed the origin of the diffraction band from liquid paraffins in terms of a cluster of seven hexagonally packed chains in the all-trans conformation. This model was confirmed by Pierce (1935) and by Zachariasen (1935) who calculated the radial distribution of chains in the liquid. These workers, however, assumed that liquid paraffin is an isotropic structure, and this is clearly not valid in the case of lipid bilayers. Calorimetric data (Chapman et al. 1967) has been used to examine the hydrocarbon chain motion above the phase transition temperature of anhydrous lecithins (Phillips et al. 1969). The heat involved in the transition is found to be about 95 % of the total heat of fusion. The total entropy per methylene group is the same for all long-

206

D. CHAPMAN

chain compounds in the crystalline form at their chain melting point. The entropy gain during the transition from ft crystal to isotropic liquid for n-alkanes, triglycerides and fatty acids is 2-6 e.u. per CH2 group, whereas for the crystal to liquid crystal transition for lecithins the equivalent figure is i • i e.u. Thus, in the liquid crystalline state the chain fluidity is about half that found in liquid n-alkanes at the transition temperatures. It was suggested that this may arise by inhibition of rotation about the carbon-carbon bonds due to the presence of the neighbouring chains (Phillips et al. 1969). The i.r. spectroscopic studies (Chapman, 1958; Chapman et al. 1966) and the Raman spectroscopic studies (Lippert & Peticolas, 1971) are consistent in showing that some rotational isomerism about the carboncarbon bonds occurs in the anhydrous and lipid-water systems. Calorimetric studies (Phillips et al. 1969) with the fully hydrated lecithins showed that they have greater chain mobility above the transition temperature than do the anhydrous or monohydrate liquid crystals but still less than that which occurs in the w-alkane melt so that some order is retained and the chains are never in a chaotic arrangement (Williams & Chapman, 1970). Below the transition temperature the area per lipid molecule is about 48 A while above this temperature (at 40 % H2O) the area per molecule is some 70 A2 (see Chapman et al. 1967; Phillips & Chapman, 1968). These dimensions are also consistent with the concept that in the lipid systems examined there is still some order normal to the plane of the membrane. The theoretical studies of Whittington & Chapman (1966) emphasized the steric repulsion of neighbouring chains which limits CH2 rotational freedom and makes such motion cooperative between these chains. Seelig (1970) used spin labels to investigate the order along hydrocarbon chains. Hubbell & McConnell (1971) also analysed the chain motions in terms of gauche-trans isomers. The recent deuterium magnetic resonance studies of Seelig & Seelig (1974) and Seelig & Niederberger (1974), comparing n.m.r. and spinlabel studies, and that of Charvolin et al. (1973) provide good evidence on the order parameter which shows that within the lipids which they examined the order parameter is essentially constant along the chain except for the region near the centre of the bilayer (see Fig. io and p. 219). It should also be noted that as the lipid is heated to higher tempera-


207

5 6 7 10 Carbon atom Fig. 10. Order parameter, at 28 °C, shown as a function of a position along the chain. Dashed lines, calculation for different values of lateral pressure (in dynes/cm); solid lines, data by Seelig & Neiderberger (after Marcelja, 1974).

tures above the transition temperature the disorder in the chain will increase (Chapman, 1958). C. Correlation with monolayer properties

Chapman (1966) and Phillips & Chapman (1968) pointed out the correlation which exists between the monolayer properties at the airwater interface of lipids and the properties of the aqueous dispersions of lipids. The condensed monolayer corresponds to the 'gel-type' structure and the expanded monolayer to the 'fluid condition' above the lipid transition temperature, so that similar thermotropic phase changes occur with the monolayers (Fig. 11). All monolayer states are possible with the saturated lecithin and phosphatidylethanolamine homologues (Phillips & Chapman, 1968). It is apparent that if the hydrocarbon chains are sufficiently long, condensed monolayers are formed whereas with shorter chains liquidexpanded films occur. These two limiting states are sufficiently well

208

D. CHAPMAN 50

40

30

S 20

10

40

60

80 Area (A/molecule)

100

120

Fig. I I . Pressure-area curves for saturated 1,2-diacylphosphatidylcholines on o-i M-NaCl at 22 °C. Q . C 2ai O, C 1 8 ; x , C 1 6 ; A , C 1 4 ; V C 10 (Phillips etal. 1968).

defined so that at any particular temperature only one of the homologues studied exhibits the transition state. The data also indicate that variations in hydrocarbon chain length which do not give rise to a change in monolayer state do not have a significant effect on the IT-A. curves. Figure 11 demonstrates how temperature changes can give rise to the above physical states in a monolayer of a single homologue. Obviously, a sufficiently low temperature causes the film to become completely condensed, whereas at higher temperatures it is fully expanded. Monolayers in the two limiting states are more or less invariant with temperature and it is only the sensitivity of the phase transition to temperature that leads to the variety of isotherms depicted in Fig. 12. The molecules in a completely condensed phosphatidylethanolamine film are much more closely packed than are those in the equivalent lecithin monolayer. This can also be correlated with the bulk properties. The lecithins have a lower transition temperature than does the equiva-


209

50 r*

40

30

8

I

20

10

40

60

80

100

120

Area (A2/molecule) Fig. 12. Pressure-area curves for 1,2-dipalmitoyl-L-phosphatidylcholine on o-i M-NaCl at various temperatures. • , 34-6°; A, z9-5°; • , 26-0°; x , 21-1°; O, i6-8°; A. 124°; D . 642° (Phillips et al. 1968).

lent chain length phosphatidylethanolamine. This presumably arises from steric factors associated with the large polar groups on the lecithin molecules. Marcelja (1974) has used this correlation between the monolayers and lipid-water dispersions to calculate the lateral pressure in the hydrocarbon chain region. He estimates this to be of the order of 20 dynes/cm for each half of the bilayer (see p. 207). Lipid phase transitions have been studied in planar lipid bilayer systems after creating domains of fluid and domains of rigid lipid (Pagano, Cherry & Chapman, 1973). D. Phase separation

The first studies on phase separation of lipid water systems were discussed by Ladbrooke & Chapman (1969) who reported studies of binary mixtures of lecithins using calorimetry (Phillips, Ladbrooke & Chapman, 1970). These authors examined mixtures of distearoyl and 14

QRB 8

210

D. CHAPMAN 60 r

50 Moles % DSL Fig. 13. Temperature composition diagrams for binary mixtures of saturated lecithins dispersed in excess (50 wt %) water, (a) i,2-distearoyl lecithin-i,2dipalmitoyl lecithin-water system (DSL-DPL), (6) 1,2-distearoyl lecithin1,2-dimyristoyl lecithin-water systems (DSL-DML). x , onset temperature from d.s.c. heating curve; I , onset temperature from d.s.c. cooling curve (Ladbrooke & Chapman, 1969).

dipalmitoyl lecithin (DSL-DPL) and also distearoyl lecithin and dimyristoyl lecithin (DSL-DML). With the DSL-DPL mixtures the phase diagram shows that a continuous series of solid solutions are formed below the Tc line. It was concluded that compound formation does not occur and that with this pair of molecules having only a small difference in chain length co-crystallization occurs (see Fig. 13). With the system DSL-DML monotectic behaviour was observed with limited solid solution formation. Here the difference in chain length is already too great for co-crystallization to occur so that as the system is cooled migration of lecithin molecules occurs within the bilayer to give crystalline regions corresponding to the two compounds (Ladbrooke &

Chapman, 1969). Examination of a series of fully saturated lecithins with dioleyl


(a)

An\

o

•a

1A

ite of heat flo

3

50

^^—80%

5oy

o2

Ai\ °

- > VU20JJ

1

/ \ioo% 10

20

(6) " Onset (cooling) I temperature,j—-—{

100 /„ DML

—"~y V-95% ' ^ > S - 90% — /

60

30 40 50 60 Temperature (°C)

mperature (°C

M

211

40 30

^

~ S* di_h_i^°''

20 -

/

/' ^c^ating)

10 0

20

40 60 80 . 100 Onset temperatures Mole % PE (heating and cooling)

Fig. 14. (a) Differential scanning calorimetry heating curves for 1,2-dimyristoyl lecithin- 1,2-dimyristoyl phosphatidylethanolamine-water mixtures, (£) phase diagram of the 1,2-dimyristoyl lecithin-1,2-dimyristoyl phosphatidylethanolamine-water mixtures (after Chapman et al. 1974).

lecithin gave similar results with phase separation of the individual components taking place (Phillips et al. 1970). Later calorimetric studies were reported by Clowes, Cherry & Chapman (1971) on mixed lecithin-cerebroside systems and on lecithin-phosphatidylethanolamine mixtures (reviewed by Oldfield & Chapman, 1972; Chapman et al. 1974). The lecithin-phosphatidylethanolamine systems of the same chain length give a wide melting range with some separation of the different lipid classes (see Fig. 14). The use of spin labels such as TEMPO to prove phase separation of mixed lipid systems has been reported by Shimshick & McConnell (1973 a) on similar lipid mixtures. Other phase separation properties have been observed by Ito & Oknishi (1974) and have shown that lipid phase separation can occur in phosphatidic acid-lecithin membranes due to the effects of Ca2+. Butler, Tattrie & Smith (1974) have shown that the probe stearic acid spin label tends to migrate to the more fluid lipid phase in multiphase systems. This confirms the earlier conclusions of Oldfield et al. (19726) and shows that measurements of membrane fluidity in heterogeneous systems are not necessarily representative of the entire membrane.

14-2

212

D. CHAPMAN

E. Cholesterol effects

In 1968 a paper was published (Ladbrooke, Williams & Chapman, 19686) describing studies on lecithin-cholesterol-water interactions by differential scanning calorimetry (d.s.c.) and X-ray diffraction. The 1,2-dipalmitoyl-L-phosphatidylcholine-cholesterol-water system was studied as a function of both temperature and concentration of components. This particular lecithin was used because it exhibits the thermotropic phase change in the presence of water at a convenient temperature (41 °C). The addition of cholesterol to the lecithin in water lowers the transition temperature between the gel and the lamellar fluid crystalline phase, and decreases the heat absorbed at the transition. No transition at all is observed with an equimolar ratio of lecithin with cholesterol in water. This ratio corresponds to the maximum amount of cholesterol which can be introduced before cholesterol precipitation occurs. These effects are not specific for dipalmitoyl lecithin; unsaturated lecithins and the lipid extract of human erythrocyte ghosts exhibit similar behaviour. The addition of cholesterol to the lecithin-water system in the gel phase causes a reduction in the cohesive forces between adjacent hydrocarbon chains of the lecithin; this leads to a fluidization of these chains. The ordered array of hydrocarbon chains in the gel phase is disrupted by cholesterol. The presence of water was also shown to be of prime importance in this interaction. The hydrocarbon chains of the lecithin in water have less thermal motion in the presence of cholesterol than they have in the absence of cholesterol above the Tc line, and they have more thermal motion than in the absence of cholesterol below the Tc line. The requirement for water has been stressed. X-ray evidence (Ladbrooke et al. 1968 A) indicates a lamellar arrangement, and at 50 % cholesterol an additional long spacing pattern occurs due to the separation of crystalline cholesterol. These results may be interpreted in terms of penetration of the lipid bilayer by cholesterol. In the lamellae of aqueous lecithin, the chains are hexagonally packed and tilted at 580. It can be envisaged that penetration will be facilitated when the chains are vertical. This causes an increase in the X-ray long spacing. At concentrations of cholesterol greater than 7-5 % the long spacing then decreases. Above this critical concentration a reduction occurs of the cohesive forces between the chains producing chain fluidization. The p.m.r. of egg-yolk lecithin shows that the addition of cholesterol produces effects which depend on


213

the state of the lipid (Chapman & Penkett, 1966). At temperatures in excess of the transition temperature, the addition of cholesterol reduces the signal due to the methylene protons of the hydrocarbon chains, i.e. the motion of these is inhibited. It has also been demonstrated that addition of cholesterol to sonicated lecithins broadens the choline residue signal to a lesser extent than it does that of the alkyl chains (Darken al. 1972). Thus the chain protons are severely immobilized. Linewidth measurements clearly demonstrate that the fluidity of the chains in the presence of cholesterol is intermediate between that of aqueous lecithin below and above its transition temperature. Even in aqueous lecithin below its transition temperature the choline group still retains appreciable mobility. Unsonicated dispersions of dipalmitoyl lecithin and cholesterol have been studied by wide-line n.m.r. spectroscopy. The observed linewidths provide a good indication of the mobility of the system. Analysis of the relative intensities of the observed signals allows the location of the steroid within the bilayer to be postulated as between the hydrocarbon chains with the steroid hydroxyl group adjacent to the phosphate group of the lecithins (Darke et al. 1972). Phase diagrams for the system DML-cholesterol-water produced from spin label studies (Shimshick & McConnell, 19736) show evidence of solid phase immiscibility but no indication of complex formation. Because of the inherent solid phase immiscibility it was possible to explain the loss of the transition when 50% cholesterol is present. When the lipid is above its transition temperature and cholesterol is added, the system passes from fluid to mixed fluid and solid, to all solid as the steroid concentration increases. There have been many other studies of cholesterol interactions with lipid systems using the spin label method (e.g. Schreier-Muccillo et al. 1973). The phase transitions of sonicated vesicles of dipalmitoylphosphatidylglycerol and dipalmitoyl lecithin in the presence and absence of cholesterol have been studied by fluorescence polarization and permeability of 22Na+ (Papahadjopouloscia/. 1973). Polarization results indicate that the probe perylene is able to penetrate the membrane interior only at temperatures in excess of the transition temperature of the lipid, and at this temperature a local maximum occurs in the diffusion rate of 22 N + . The absence of such changes on addition of cholesterol indicates that this removes the gross phase transition. Cogan et al. (1973) have used fluorescent probes to study this transition. Phillips & Finer (1974) have discussed the variation with composition

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of the cooperativity of the lipid thermotropic transition in mixed dipalmitoyl lecithin cholesterol bilayers. In bilayers containing less than equimolar amounts of cholesterol discrete regions of i: i complex separates out leaving lecithin molecules at the boundaries of these regions for which cooperative motions are not possible and clusters of free lecithin molecules which freeze at the normal transition temperature. Phillips & Finer comment on suggestions of Rothman & Engelman (1972) concerning 2:1 lipid-cholesterol complexes and also some calorimetric studies of Hinz & Sturtevant (1972). F. Metal ion effects Metal ion interactions have been known for some years to affect the thermotropic phase transition of soap systems. The thermotropic phase transition of stearic acid occurs at 114 °C with the sodium salt and at 170 °C with the potassium salt. These phase transitions can be linked to the monolayer characteristics. Early studies of stearic acid monolayers (Harkins & Anderson, 1937; Shanes & Gershfeld, i960) showed that interaction with Ca2+ ions caused an increase in surface pressure (i.e. condensation) and also decreased the permeability to water. The same effect has been observed with phosphatidylserine monolayers (Rojas & Tobias, 1965), but Na+ and K+ addition gave no such condensation. Later, more extensive studies showed that a variety of acidic phospholipid monolayers undergo an increase in surface potential and decrease in surface pressure on addition of Ca2+ and other bivalent cations (Papahadjopoulos, 1968). Phosphatidylserine is found to be more selective than phosphatidic acid but for both systems the order of cation effectiveness is Ca2+ > Ba2+ > Mg2+. The formation of linear polymeric complexes was proposed to account for these findings. Cationic charge has been observed to be important in some bilayer studies of phosphatidylserine (Ohki, 1969). Black membranes formed in the presence of Ca2+ ions are found to be more stable with a higher electrical resistance than those formed in the presence of Na + only. The concentrations of cationic species required to produce charge reversal in phosphatidylserine dispersions have been determined together with association constants for the species formed (Barton, 1968). The results obtained agree well with those obtained previously


215

(Blaustein, 1967) with the exception of uranyl cation UO| + . Recent studies with this cation (Chapman et al. 1974) indicate that this ion causes the thermotropic phase transition temperature of lecithins to increase. Two main phase transitions were observed corresponding to the presence of complexed and uncomplexed lipid. When the titration is complete only the higher melting transition remains. The studies by Chapman et al. (1974) indicate that the interaction between cations and phosphatidylserine causes greater shifts of transition temperature than is observed with lecithin molecules. All the cations studied were found to shift the phase transition temperature of the phospholipids to higher values. The precise nature of the interaction between ions and phospholipids is still open to doubt. There is some evidence that charge neutralization is the prime interaction of charged phospholipids with divalent cations (Verkleij et al. 1974; Trauble & Eibl, 1974). Divalent cations were found to increaase the transition temperature and the monovalent cations to fluidize the bilayer. Some authors believe that the primary effect of the cation on lecithins may be on the aqueous portion of the lipid bilayer (Gottlieb & Eanes, 1972; Ehrstrom et al. 1973; Godin & Ng, 1974). A recent study of an extensive range of salts with lecithin bilayers (Chapman et al. 1974) indicates that the anion present has a very large effect in determining the state of fluidity of the bilayer, the results obtained being best explained by a thermodynamic treatment based on relative association constants. Trauble (1971) has shown that pH can affect lipid transition temperatures, particularly lipids such as the phosphatidylethanolamines. Verkleij et al. (1974) have shown that the thermotropic phase transition of a synthetic phosphatidylglycerol is influenced by pH, Ca2+, and a basic protein of myelin. G. Protein and polypeptide effects

Various studies have now been made of the effects of polypeptides and proteins on phase transition temperatures. Chapman et al. (1974) have studied phosphatidylserine interactions with cytochrome C lysozyme and polylysine and have shown shifts of transition. The 'electrostatic' effect of polypeptides and proteins on lipid fluidity may also be important in transient and structural situations. It is clearly relevant where comparisons are made between transitions which occur with membranes where electrostatic interaction of lipid and protein is thought to occur, compared to those of the extracted lipids.

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D. CHAPMAN

This effect on lipid fluidity by interaction of protein with the polar groups of lipids may also occur in other important systems, such as the concanavalin A-glycolipid system and antigen-antibody interactions and could be of considerable biological importance. Gramicidin A is an ionophore which is thought to transport ions by means of a pore mechanism (Urry, 1971; Hladky & Haydon, 1972). With this molecule, the pretransitional peak is affected at low polypeptide concentrations, suggesting that the packing of the lecithin polar groups has been affected, but in addition to this the heat involved in the main lipid endothermic transition is markedly reduced in a somewhat similar manner to that observed with cholesterol. It seems reasonable to conclude, therefore, that as with cholesterol the molecule is interdigitated among the lipid chains preventing chain crystallization from occurring. Consistent with this it is postulated that gramicidin A forms channels bridging the lipid bilayers. This result is also interesting in view of the results on black lipid membrane systems. Krasne, Eisenman & Szabo (1971) have shown that gramicidin A was able to mediate potassium ion transport above and below the transition temperature of the lipid forming a' black lipid film'. (It is possible that whilst the bulk of the lipid below the transition temperature was rigid, the lipid immediately adjacent to the gramicidin A was fluid.) Nonactin and valinomycin are observed to act as ion carriers (Shemyakin et al. i960) and so must be able to diffuse freely throughout a lipid bilayer. The effectiveness of the ion carriers is found to decrease dramatically below the transition temperature of the lipid systems studied (Krasne et al. 1971). A more detailed study of the effects of valinomycin on lecithin bilayers using Fourier Transform and pulsed p.m.r. spectroscopy has shown that 0-2 mole% of the drug lowers the transition temperature of DPL by ~ 1°; the transition is also found to occur over a wider temperature range than in the absence of the drug (Hsu & Chen, 1973). Chapman & Urbina (1971) suggested that lipid chain crystallization can cause protein molecules to be squeezed out of that portion of the bilayer. Consistent with this idea is the evidence that lipid phase separation can cause protein aggregation (Speth & Wunderlich, 1973). When alveolar membranes are grown at 28 °C the component protein molecules are randomly distributed, whereas chilling the membrane to 5 °C causes aggregation.

Phase transitions and fluidity characteristics 217 H. Drug interactions

The precise biological and pharmacological actions of drug molecules are largely undetermined. There is, however, widening belief that interaction with the lipid constituents of membranes resulting in an alteration of the fluidity characteristics may be an important mode of action of the drugs. There are now available a number of studies showing that drug molecules can affect the thermotropic phase transition of lipid-water systems. Sometimes this corresponds to a removal of the transition somewhat similar to that observed with cholesterol. We can instance gramicidin A (Chapman et al. 1974) and other antibiotic molecules (Pache, Chapman & Hillaby, 1972). On the other hand, sometimes the drug shifts (with increasing concentration) the lipid transition temperature to lower values, and we can instance a range of antidepressant drug molecules, e.g. desipramine (Cater et al. 1974). Hill (1974) has studied the effects of a series of normal alcohols (up to C8) as well as three inhalation anaesthetics on the lipid phase transition temperatures and has shown shifts both to lower and higher values. Trudell, Hubbell & Cohen (1973) have studied the effects of inhalation anaesthetics on the order of nitroxide-substituted egg-yolk lecithin bilayers to predetermine whether any observed effects could be extrapolated to biological systems. It was observed that the anaesthetics studied produced a more disordered bilayer, the degree of disorder being linearly dependent on dose within the concentration range employed for clinical anaesthesia. In addition, high pressures of helium were found to antagonize the effect of the anaesthetic paralleling the known pressure reversal effect observed with these substances (Johnson & Miller, 1970). Trauble (1972) has studied the dye molecule bromothymolblue, a pH indicator of a pH of 7 1 . A marked decrease in the absorption band at A = 615 nm is observed when lipid is added to an aqueous solution of bromothymolblue at pH 7. The extinction coefficient EG15 of the membrane-bound species of bromothymolblue (BTB~ or/and BTP°) is more than two orders of magnitude smaller than the extinction coefficient of bromothymolblue in an aqueous solution of pH 7 (2?615 = i'4xio 4 ). The phase transition is therefore accompanied by a sharp decrease in the optical density. He also carried out titrations of dipalmitoyl lecithin dispersions with i-anilino-8-naphthalenesulphonate (ANS) at temperatures below and above Tt. These experiments show

2l8

D. CHAPMAN

that the increase in ANS fluorescence intensity at the phase transition is mainly due to an increase in the number of binding places for ANS on the membrane surface. Many studies of drug interactions on lipid transitions are to be expected. It may be that studies of drug interactions of this sort will be useful as a method of indicating broad ranges of pharmacological action (e.g. antidepressant action, see Cater et al. 1974). I. Permeability characteristics

Studies have been made of permeability characteristics for water and various other molecules above and below the lipid phase transition temperature; e.g. the kinetics of water permeability through lipid systems has been studied and related to lipid fluidity (Bittman & Blau, 1972). Thus there is a marked increase in water permeability as the lipid chains become more unsaturated. This is also the case with non-electrolytes such as glycerol and erythritol (de Gier, Mandersloot & van Deenen, 1968). The self-diffusion rate of 22Na+ through lecithin bilayers also shows a marked increase at the transition temperature to the liquid crystalline form (Papahadjopoulos et al. 1973). The effect of cholesterol on the lipid chains when the lipid is in the fluid condition is to decrease water permeability (Bittman & Blau, 1972). Black lipid membranes also show the same effect (Finkelstein & Cass, 1967). These results are consistent with a reduction in the fluidity characteristics of the lipid chains. On the other hand, the presence of cholesterol is to enhance the rate of water permeability of liposomes derived from saturated liposomes (Bittman & Blau, 1972). Inoue (1974) has studied the permeability characteristics of liposomes to glucose. It is suggested that the permeability of glucose through vesicles of DPL and DML is enhanced drastically at their transition temperatures. Nicholls & Miller (1974) have shown that liposomes of these lipids are more permeable to chloride ions above their transition temperatures than are egg-yolk liposomes. J. Theoretical treatments

A number of theoretical studies have now been made of the thermotropic phase transition of lipids and lipid-water systems. Whittington & Chapman (1965) examined the motion of chains in the hexagonal form of long-chain molecules. In later studies they also examined (1966) the way in which the end-to-end distance of chains (which are fixed


219

at one end) varies as the distance between the chains is increased. They used two different potential functions and restricted the chains to a two-dimensional hexagonal lattice. This study emphasized the cooperative nature of the twisting and movement of the CH2 groups of adjacent chains leading to the phase transition and also above it in the more fluid state. Rothman (1973) and Nagle (1973) have also emphasized this cooperative nature of chain movements. Marcelja (1974) has used a molecular field approximation and examined different statistical averages over all conformations of a single chain in a field due to neighbouring molecules. This method has been extended to describe the properties of lipid monolayers and bilayers. The theoretical pressure-area diagrams are in good agreement with the experimental data of Phillips & Chapman (1968). The order parameter for hydrocarbon chains as a function of temperature, lateral pressure and position along the chain is also calculated and compared with the experimental. A comparison between calculated and experimental order parameters (using n.m.r. methods) is shown in Fig. 10. This shows the order parameter at 28 °C as a function of a position along the chain for different values of lateral pressure. Temperatures and latent heats of the thermal phase transition were also calculated and are of the correct magnitude (Marcelja, 1974). III.

T H E R M O T R O P I C PHASE TRANSITIONS OF CELL MEMBRANES

There is now very strong evidence that the lipid bilayer forms an important part of naturally occurring membranes (Wilkins, Blaurock & Engelman, 1971); the lipid packing may be heterogeneous and contain both fluid and rigid regions (Oldfield & Chapman, 1972). Model systems have been previously discussed (Phillips et al. 1970; Chapman et al. 1974) and remarkably similar behaviour is observed with some natural membranes. A. Myelin membranes

The first cell membranes to show thermotropic lipid phase transitions were those of ox-brain myelin and myelin of human origin. This study showed that lipid phase transitions did occur but only after some dehydration occurred. Thus freeze-dried myelin exhibits endotherms when examined using a differential scanning calorimeter (Ladbrooke

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et. al. 1968 a). It was shown that this is associated with a precipitation of cholesterol due to dehydration below a certain water content (some 20%). With wet fresh myelin no lipid thermal transitions are observed. This can be associated with the presence of the cholesterol in the membrane keeping the lipid in the intermediate fluid-type condition. In the absence of cholesterol the myelin lipids can crystallize at body temperature. B. Erythrocyte membranes The erythrocyte membrane was studied in 1968 using calorimetry. Preliminary results indicated that there were similarities with myelin in that cholesterol inhibits the crystallization of the polar lipids and for this effect there is a requirement for bound water. A difficulty with this membrane is that transitions due to the lipid were significantly smaller than with myelin. The membranes exhibited a broad endotherm when cholesterol was removed (Ladbrooke et al. 1968 a). C. Acholeplasma laidlawii membranes Acholeplasma laidlawii membranes show a marked endotherm when examined using calorimetry. It occurs at the same temperature as does that of the extracted lipids dispersed in water (Steim et al. 1969). Supplemented and unsupplemented Acholeplasma laidlawii exhibit phase transitions similar to those of mixed lipid systems (Steim et al. 1969; Chapman & Urbina, 1971; Oldfield, Chapman & Derbyshire, 1972). For each system the transition observed is very wide (~ 30°) and the isolated lipids produce a very similar transition. In unsupplemented media, the transition range encompasses the growth temperature, hence at this temperature the ordered hydrocarbon gel state predominates; this phase is found to disappear only at temperatures well above that of growth (Engelman, 1971). Engelman showed that the hydrocarbon chains in the palmitate-enriched membranes exhibit an area per lipid molecule of 43 A2 whilst above the transition temperature the area is found to be some 60 A2. Deuteron magnetic resonance studies of these cells supplemented with perdeuterated fatty acids indicated the presence of extensive rigid lipid regions at the growth temperature of this organism (Oldfield et al. 1972 a). Normally, Acholeplasma laidlawii contains no lecithin molecules but this can be readily incorporated in large amounts (Grant & McConnell, 1973) into the membranes. The viability of the mem-


221

brane is unaffected by this added phospholipid which is found to diffuse through the membrane. The transition temperature of the lecithin is found to drop, hence its fluidity is increased and the observed transition is broadened because of lateral phase separation of regions relatively rich in lecithin. Discussions of the possible relationships which occur between the lipid-phase melting temperature and discontinuities in the Arrhenius plots of ATPase enzyme activity have been given. In these systems the discontinuity usually occurs at or below this transition temperature (Verkleij et al. 1972). D. Escherichia coli membranes The growth medium of E. coli is found to have marked effects on the thermal transition observed. When the fatty acid composition of the medium is altered the observed transition temperature shifts as in simple lipid-water systems; this is also observed with Acholeplasma laidlawii (Esfahani et al. 1971). For both systems, Arrhenius plots of growth rates are found to depend on the fatty acids in the growth medium (Fox & Tsukagoshi, 1972). The growth temperature of E. coli is 37 °C and this is encompassed by the observed thermal transition. Elaidatesupplemented membranes have a lower transition temperature than do linolenate or myristoleate-supplemented; thus alteration of the relative concentrations of fatty acids in the growth medium provides a mechanism for controlling the fluidity of the membrane. Uptake of sugars by E. coli is found to be dependent on the fatty acid composition of the bacteria (Overath, Schairer & Stoffel, 1970). The rate of uptake is found to change abruptly at the temperature where lateral diffusion of the phospholipids present becomes rapid enough to allow mixing to occur. The transport properties of E. coli are very dependent of the fatty acid supplements (Linden, Keith & Fox, 1973). Arrhenius plots exhibit a biphasic shape, and the temperatures at which the plots change slope reflect the melting characteristics of the supplement, i.e. depend on the length and degree of unsaturation of the hydrocarbon chains. These temperatures correspond to those of the phase changes of the supplement. There is also evidence (Linden et al. 1973) that two characteristic temperatures occur, corresponding to the upper and lower temperature limits of phase separation of the lipids in the membrane. At temperatures below the lower limit, the activation energy for transport is found to be very large; as the upper limit is reached, this parameter decreases and approaches zero. In the former instance all the component lipids are

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I

I

1-2 31

3-3

3-4

3-6

lfKxlO3 Fig. i s. A suspension of inner membranes of a fatty acid auxotroph of E. coli cells (5 mg protein per ml) derived from linoleate-grown cells was labelled with 6N11 spin label. The rates of the mid-field and high-field line heights are plotted as a function of the reciprocal of the absolute temperature (after Linden et al. 1973).

in the solid phase and so transport is difficult. At the upper limiting temperature the increased fluidity of the lipids present results in an increase in their isothermal lateral compressibility and so transport becomes easier and the observed decrease in activation energy occurs. A diagram illustrating the phase transitions detected using spin labels (6N11) with the E. coli auxotroph is shown in Fig. 15. E. Yeast membranes Mitochondrial membranes from cells of Saccharomyces cerevisiae grown under anaerobic and aerobic conditions and supplemented and unsupplemented with fatty acids have been examined using a variety of


223

physical techniques (calorimetry, X-ray, spin labels, and light scattering) (Bertoli, Chapman & Strach, 1974) which show considerable differences in the lipid endotherms obtained with the different cells. The anaerobic unsupplemented membrane exhibit a transition which begins above the growth temperature and extends to some 70 °C whilst the aerobic unsupplemented cells exhibit a transition extending from - 2 0 to 10 °C. F. Other membranes

A number of other cell membrane systems have been studied (Blazyk & Steim, 1972). These include rat liver mitochondria and microsomes which also show wide thermal transitions associated with their lipid components. Particle aggregation induced by lipid phase transitions have also been observed in Streptococcus faecalts membranes but not in Staphylococcus aureus membrane systems (Haest et al. 1974). In experiments with sweet potato mitochondria, Raison, Lyons, & Thomson (1971) found that the Arrhenius plot for succinoxidase activity has a break at 9-10 °C. However, if the mitochondria are first treated with detergent, which brings the mitochondrial lipids into solution or alters their physical arrangement in the membranes, then the Arrhenius plot becomes linear; in other words, activation energy is then uniform over the whole temperature range. The authors conclude that a break in the Arrhenius plot, indicating a temperature-dependent change in activation energy, is associated with a change in the physical state of the lipids, which in turn influences the configuration of enzyme proteins (such as succinoxidase) that are embedded in the membrane. Comparative studies of mitochondria from the livers of a variety of animals show that it is only in homeotherms such as the rat that the Arrhenius plot for mitochondrial enzymes is non-linear (Lyons & Raison, 1970); fish liver mitochondria yield linear Arrhenius plots with no phase transition. It has been suggested that lipids adjacent to a protein moiety in a membrane have a considerable difference of fluidity and different from that of the rest of the lipid present. Stier & Sackmann (1973) suggest that liver microsomal membranes contain a rigid halo of lipids in semicrystalline form enclosing pockets of cytochrome enzyme, whilst the remaining portion of lipid is in a fluid state. This idea is an interesting one in that it shows how an enzyme might be affected by a lipid phase transition from only a small portion of the lipid present in the membrane.

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IV. THE SIGNIFICANCE OF LIPID PHASE TRANSITIONS FOR BIOLOGICAL SYSTEMS

We can summarize the various changes which occur at the transition temperature in the lipid-water systems: (a) An expansion of the lattice and a decrease in bilayer thickness. (b) Increased rotational isomerism of CH2 groups about C—C bonds. (c) Increased mobility of N(CH3)3 groups. (d) Increased diffusion rate of lipids above the transition temperature. (e) Some change in bound water interactions at the transition temperature. We also know that the thermotropic transition can be shifted by interaction with metal ions, pH, polypeptide or protein interactions (p.. 215). Furthermore, the permeability characteristics for various molecules, e.g. water, and also the possibility of molecules penetrating or partitioning into the membrane are also dependent upon whether the lipid is below or above its transition temperature. A. Fluidity The question is sometimes asked - As biological systems are remarkably constant in temperature, are these particular lipid phase transitions important for biological systems? It seems clear from an examination of the various lipid-water phase diagrams that with a biological system at constant temperature where there is a bilayer region of lipid forming the membrane the degree of ' fluidity' is a direct reflexion of the transition temperature of the lipids; i.e. the fluidity is greatest when highly unsaturated lipids are present and less when more saturated lipids are present. (There can be modulating effects of cholesterol, proteins, etc.) In some cases the transition temperature of the lipids will be below, or at growth temperature; in other cases it may be above the growth temperature or encompassing it (Oldfield & Chapman, 1972). The appropriate phase transition of lipids will therefore determine the correct fluidity of a cell membrane and also the correct lipid phase separation characteristics (as in Acholeplasma laidlawii membranes). These in turn can affect membrane elasticity, the insertion, aggregation and diffusional movements of the protein and lipid components as well as permeability characteristics. Where we have a membrane that contains cholesterol


225

(e.g. erythrocyte or myelin membranes) and the cholesterol is able to move into and out of the membrane it is possible for lipid phase separation to occur so that the lipid in that region will spontaneously crystallize out, thus changing the local permeability properties. (Another possibility that exists but for which there is little experimental evidence as yet is that in some cases there may be asymmetry of fluidity of bilayer membranes. *) B. Trigger mechanisms

At constant temperature triggering mechanisms may occur as a result of interactions with the membrane by metal ions, proteins or drugs so that local changes of fluidity, phase separation and hence changes of permeability characteristics may also take place as a result of such interactions. Trigger mechanisms of this type could be particularly important, leading to lateral information transfer along the cell membrane. Other processes involving the cell have been linked to the lipid phase transitions, including cell fusion and enzymatic activities (Papahadjopoulos et al. 1974). Fusion processes have been shown to be related to the phase transitions. Lipids above their transition temperatures are found to be more susceptible to fusion. The presence of cholesterol in lipids which were above their phase transition temperature reduced fusion processes. In more speculative vein suggestions have been made linking the temperature-dependence of the action potential in Nitella flexilis with lipid phase transitions of the membranes of these systems (Blatt, 1974). Further speculations (Trauble & Eibl, 1974) have been made linking the effects of metal ions on lipid phase transitions with recent ideas about the mechanism of nerve excitation where abrupt cooperative conformational changes occur in the axonal membrane during excitation. The possibility of cations directly affecting the structure of presynaptic or axonal membranes to enhance or reduce nerve activity by affecting the structure of presynaptic or axonal membranes to enhance or reduce nerve activity by affecting lipids was also suggested. Not all biological systems operate at a single constant temperature. Poikilothermic organisms adapt themselves to their environmental temperature and the lipid composition of their cell membranes changes with this temperature. The lipids of these membranes become more • Where lipid class asymmetry occurs if the same fatty acids are present this could give rise to asymmetric lipid fluidity. 15

QRB

8

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unsaturated at lower environmental temperature and more saturated at higher environmental temperatures. It has been suggested that this operates through a feedback mechanism to retain a constant fluidity of the membranes so as to keep diffusional and permeability processes at the correct optimum (Chapman, 1966). C. Cryobiology

In certain practical situations it is important to freeze cells and tissues, as in the various requirements in the field of cryobiology, so that major changes of temperature are involved. When cells or membranes are frozen to very low temperatures, lipid phase characteristics and phase separation need to be considered. Whether the lipid crystallizes before the ice melting point or at a lower temperature could be important in determining freeze damage. The various phenomena of lipid phase separation and protein aggregation need to be considered from this point of view. V. ACKNOWLEDGEMENTS

I wish to thank Dr B. Kingston for collecting some of the information used in this review, and J. Schechenzuber and V. Kovacic, of the Biophysics Department of the Pennsylvania State University, University Park, Pennsylvania (U.S.A.), for typing the manuscript.

VI.

REFERENCES

R. W., BELL, J. D., RADDA, G. K. & RICHARDS, R. E. (1972). Phosphorus nuclear magnetic resonance in phospholipid dispersions. Bio-

BARKER,

chitn. biophys. Ada 260, 161-3. M. D., GREEN, D. K. & CHAPMAN, D. (1969). E.s.r. studies of nitroxide probes in lecithin-water systems. Chem. Phys. Lipids 3, 140-4. BARTON, P. G. (1968). The influence of surface charge density of phosphatides on the binding of some cations. J. biol. Chem. 243, 3884-90. BEAR, R. S., PALMER, K. J. & SCHMITT, F. O. (1941). X-ray diffraction studies of nerve lipids. J. cell. comp. Physiol. 17, 355-67. BERNAL, J. D. (1933). General discussion. Trans. Faraday Soc. 29, 1082. BERTOLI, E., CHAPMAN, D. & STRACH, S. J. (1975). Biomembrane phase transitions. Studies of Saccharomyces cerevisiae. (In the Press.) BITTMAN, R. & BLAU, L. (1972). The phospholipid-cholesterol interaction. Kinetics of water permeability in liposomes. Biochemistry, N.Y. n , 4831-9. BARRATT,


227

F. J. (1974). Temperature dependence of the action potential in Nitella flexilis. Biochim. biophys. Ada 339, 382-9. BLAUSTEIN, M. P. (1967). Phospholipids as ion exchangers: Implications for a possible role in biological membrane excitability and anesthesia. Biochim. biophys. Ada 135, 653-68. BLAZYK, J. F. & STEIM, J. M. (1972). Phase transitions in mammalian membranes. Biochim. biophys. Acta 266, 737-41. BUTLER, K. W., TATTRIE, N. H. & SMITH, I. C. P. (1974). The location of spin probes in two phase mixed lipid systems. Biochim. biophys. Acta 363, 351-60. BYRNE, P. & CHAPMAN, D. (1964). Liquid crystalline nature of phospholipids. Nature, Lond. 202, 987-8. CATER, B. A., CHAPMAN, D., HAWES, S. & SAVILLE, J. (1974). Lipid phase transitions and drug interactions. Biochim. biophys. Acta 363, 54-69. CHAPMAN, D. (1958). An infrared spectroscopic examination of some anhydrous sodium soaps. J. chem. Soc. 152, 784-9. CHAPMAN, D. (1962). The polymorphism of glycerides. Chem. Rev. (5), 433-56. CHAPMAN, D. (1966). Liquid crystals and cell membranes. Ann. N.Y. Acad. Set. 137, 745-54. CHAPMAN, D. (1971). Liquid crystalline properties of phospholipids and biological membranes. Symp. Faraday Soc. (5), 163-74. CHAPMAN, D. (1972). Nmr spectroscopic studies of biological membranes. Ann. N.Y. Acad. Sci. 195, 179-206. CHAPMAN, D., BYRNE, P. & SHIPLEY, G. G. (1966). The physical properties of phospholipids. I. Solid state and mesomorphic properties of some 2,3-diacyl-DL-phosphatidylethanolamines. Proc. R. Soc. A 290, 11542. CHAPMAN, D. & CHEN, S. (1972). Thermal and spectroscopic studies of lipids and membranes. Chem. Phys. Lipids 8, 318-26. CHAPMAN, D. & COLLIN, D. T. (1965). Differential thermal analysis of phospholipids. Nature, Lond. 206, 189. CHAPMAN, D. & PENKETT, S. A. (1966). Nmr spectroscopic studies of the interaction of phospholipids with cholesterol. Nature, Lond. 211, 13045CHAPMAN, D. & SALSBURY, N. J. (1966). Physical studies of phospholipids. V. Proton magnetic resonance studies of molecular motion in some 2,3diacyl-DL-phosphatidylethanolamines. Trans. Faraday Soc. 62, 2607BLATT,

21. CHAPMAN,

D. & URBINA, J. (1971). Phase transition and bilayer structure of Mycoplasma laidlawii B. FEBS Lett. 12(3), 169-72. CHAPMAN, D., URBINA, J. & KEOUGH, K. M. (1974). Biomembrane phase transitions. Studies of lipid-water systems using differential scanning calorimetry. J. biol. Chem. 249, 2512-21. CHAPMAN, D., WILLIAMS, R. M. & LADBROOKE, B. D. (1967). Physical studies of phospholipids. VI. Thermotropic and lyotropic mesomorphism of 15-2

228

D. CHAPMAN

some 1,2-diacylphosphatidylcholines (lecithins). Chem. Phys. Lipids i, 445-75CHARVOLIN, J., MANNEVILLE, P. & DELOCHE, B. (1973). Magnetic resonance of perdeuterated potassium laurate in oriented soap-water multilayers. Chem. Phys. Letters 23, 345-8. CLOWES, A., CHERRY, R. J. & CHAPMAN, D. (1971). Physical properties of lecithin-cerebroside films. Biochim. biophys. Acta 249, 301-7. COGAN, U., SHINITZKY, M., WEBER, G. & HISHIDA, T. (1073). Microviscosity and order in the hydrocarbon region of phospholipid and phospholipidcholesterol dispersions determined with fluorescent probes. Biochemistry, N.Y. 12, 521-7. DARKE, A., FINER, E. G., FLOOR, A. G. & PHILLIPS, M. C. (1972). Nuclear magnetic resonance study of lecithin-cholesterol interactions. J. molec. Biol. 63, 265-78. DE GIER, J., MANDERSLOOT, J. G. & VAN DEENEN, L. L. M. (1968). Lipid composition and permeability of liposomes. Biochim. biophys. Acta 150, 666-75. DERVICHIAN, D. G. (1964). The physical chemistry of phospholipids. In Progress in Biophysics and Molecular Biology 14, 263-342. New York: Pergamon Press. DEVAUX, P., SCANDELLA, C. J. & MCCONNELL, H. M. (1973). Spin-spin interactions between spin labelled phospholipids incorporated into membranes. J. Am. chem. Soc. 95, 474-85. EHRSTROM, M.,

ERIKSONN, L. E. G.,

ISRAELACHVILI, J. & EHRENBERG, A.

(1973). The effects of some cations and anions on spin-labelled cytoplasmic membranes of Bacillus subtilis. Biochem. biophys. Res. Commun. 55. 396-402. ENGELMAN, D. M. (1971). Lipid bilayer structure in the membrane of Mycoplasma laidlawii. J. molec. Biol. 58, 153-65. ENGELMAN, D. M. & ROTHMAN, J. E. (1972). The planar organization of lecithin-cholesterol bilayers. Proc. natn. Acad. Set. U.S.A. 247, 3694-5. ESFAHANI, M.,

LIMBRICK, A. R.,

KNUTTON, S., OKA, T.

& WAKIL, S. J.

(1971). The molecular organization of lipids in the membrane of Escherichia coli. Proc. natn. Acad. Set. U.S.A. 68, 3180-4. FINER, E. G. & DARKE, A. (1974). Phospholipid hydration studied by deuteron magnetic resonance spectroscopy. Chem. Phys. Lipids 12, 1-16. FINEAN, J. B. (1953). X-ray diffraction study on the polymorphism of phospholipids. Biochim. biophys. Acta 10, 371-84. FINEAN, J. B. & MILLINGTON, P. F. (1955). Low angle X-ray diffraction study of the polymorphic forms of synthetic a:/? and a:a' kephalins and a:ft' lecithins. Trans. Faraday Soc. 51, 1008-15. FINKELSTEIN, A. & CASS, A. (1967). Effect of cholesterol on the water permeability of thin lipid membranes. Nature, Lond. 216, 717-18. Fox, F. C. & TSUKAGOSHI, T. (1972). The influence of lipid phase transitions on membrane function and assembly. In Membrane Research, p. 145. New York: Academic Press.


229

D. V. & NG, T. W. (1974). Studies on the membrane-perturbational effects of drugs and divalent cations utilising trinitro benzene s sulfonic acid. Mol. Pharmacol. 9, 802-19. GOTTLIEB, M. H. & EANES, E. D. (1972). Influence of electrolytes on the thickness of the phospholipid bilayers of lamellar smectic mesophases. Biophys. J. 12, 1533-48. GRANT, C. W. M. & MCCONNELL, H. M. (1973). Fusion of phospholipid vesicles with viable Acholeplasma laidlawii. Proc. natn. Acad. Set. U.S.A. 70, 1238-40. GULIK-KRZYWICKI, T., SCHECTER, E., LUZZATI, V. & FAURE, M. (1969). Interactions of proteins and lipids: Structure and polymorphism of protein-lipid-water phases. Nature, Lond. 223, 1116-21. GODIN,

HAEST, C. W. M., VERKLEIJ, A. J., DE GIER, J., SCHEEK, R., VERVERGAERT, P. H. J. & VAN DEENEN, L. L. M. (1974). The effect of lipid phase

transitions on the architecture of bacterial membranes. Biochitn. biophys. Acta 356,17-26. HARKINS, W. D. & ANDERSON, T. F. (1937). A simple accurate film balance of the vertical type for biological and chemical work and a theoretical and experimental comparison with the horizontal type. II. Tight packing of a monolayer by ions. J. Am. chem. Soc. 59, 2189-97. HILL, M. W. (1974). The effect of anesthetic-like molecules on the phase transition in smectic mesophases of dipalmitoyl lecithin. I. The normal alcohol up to c = 9 and three inhalation anesthetics. Biochim. biophys. Acta 356, 117-124. HINZ, H. J. & STURTEVANT, J. M. (1972). Calorimetric investigation of the in-

fluence of cholesterol on the transition properties of bilayers formed from synthetic L-a-lecithins in aqueous suspension. J. biol. Chem. 247, 3697-701. HLADKY, S. B. & HAYDON, D. A. (1972). Ion transfer across lipid membranes in the presence of gramicidin A. Biochim. biophys. Acta 274, 294-312. HORWITZ, A. F., HORSTEY, W. J. & KLEIN, M. P. (1972). Magnetic resonance studies on membrane and model membrane systems: Proton magnetic relaxation rates in sonicated lecithin dispersions. Proc. natn. Acad. Sci. U.S.A. 69, 590-3. Hsu, M. & CHEN, S. I. (1973). Nuclear magnetic resonance studies of the interaction of valinomycin with unsonicated lecithin bilayers. Biochemistry, N.Y. 12, 3872-6. HUBBELL, W. L. & MCCONNELL, H. M. (1971). Molecular motion in spinlabelled phospholipids and membranes. / . Am. chem. Soc. 93, 314-26. INOUE, K. (1974). Permeability properties of liposomes prepared from dipalmitoyl lecithin, dimyristoyl lecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin. Biochim. biophys. Acta 339, 390-402. 2+ ITO, T. & OHNISHI, S. (1974). Ca induced lateral phase separations in phosphatidic acid-phosphatidylcholine membranes. Biochim. biophys. Acta 352, 29-37. JOHNSON, S. M. & MILLER, K. W. (1970). Antagonism of pressure and anaesthesia. Nature, Lond. 328, 75-6.

230

D. CHAPMAN

W. & MCCONNELL, H. M. (1974). Lateral phase separations in Escherichia coli membranes. Biochim. biophys. Ada 345, 220-30. KORNBERG, R. D. & MCCONNELL, H. M. (1971). Lateral diffusion of phospholipids in a vesicle membrane. Proc. natn. Acad. Sci. U.S.A. 68, 2564-8. KRASNE, S., EISENMAN, G. & SZABO, G. (1971). Freezing and melting of lipid bilayers and the mode of action of nonactin, valinomycin and gramicidin. Science, N.Y. 174, 412-15. LADBROOKE, B. D. & CHAPMAN, D. (1969). Thermal analysis of lipids, proteins and biological membranes. Chem. Phys. Lipids 3, 304-67. KLEEMAN,

LADBROOKE, B. D., JENKINSON, T. J., KAMAT, V. B. & CHAPMAN, D. (1968a).

Physical studies of myelin. I. Thermal analysis. Biochim. biophys. Ada 164, 101-9. LADBROOKE, B. D., WILLIAMS, R. M. & CHAPMAN, D. (19686). Studies on lecithin-cholesterol-water interactions by differential scanning calorimetry and X-ray diffraction. Biochim. biophys. Ada 150, 333-40. LAWRENCE, A. S. C. (1938). The metal soaps and the gelation of their paraffin solutions. Trans. Faraday Soc. 34, 660-77. LEVINE, Y. K. (1973). X-ray diffraction studies of membranes. Progr. Surface Sci. 3, 279-352. 13 LEVINE, Y. K., BIRDSALL, N. J. M., LEE, A. G. & METCALFE, J. C. (1972). C nuclear magnetic resonance relaxation measurements of synthetic lecithins and the effect of spin-labelled lipids. Biochemistry, N.Y. 11, 1416-21. LEVINE, Y. K. & WILKINS, M. H. F. (1971). Structure of oriented lipid bilayers. Nature New Biol. 230, 69-72. LINDEN, C. D., KEITH, A. D. & Fox, C. F. (1973). Correlations between fatty acid distribution in phospholipids and the temperature dependence of membrane physical state. J. Supramol. Struct, i, 523-34. LIPPERT, J. L. & PETICOLAS, W. L. (1971). Laser Raman investigation of the effect of cholesterol on conformational changes in dipalmitoyl lechithin multilayers. Proc. natn. Acad. Sci. U.S.A. 68, 1572-6. LIPPERT, J. L. & PETICOLAS, W. L. (1972). Raman active vibrations in long chain fatty acids and phospholipid sonicates. Biochim. biophys. Ada 282,8-17. LUZZATI, V. (1968). X-ray diffraction studies of lipid-water systems in biological membranes. In Biological Membranes (ed. D. Chapman), p. 71. New York: Academic Press. LUZZATI, V. & HUSSON, F. (1962). The structure of the liquid crystalline phases of lipid-water systems. J. Cell. Biol. 12, 207-19. LUZZATI, P. V., MUSTACCHI, H., SKOULIOS, A. & HUSSON, F. (i960). La structure des colloides d'association. I. Les phases liquide-crystallines des systemes amphiphile-eau. Ada crystallogr. 13, 660-7. LYONS, J. M. & RAISON, J. K. (1970). A temperature-induced transition in mitochondrial oxidation contrasts between cold and warm-blooded animals. Comp. Biochem. Physiol. 37, 405-11. MARCELJA, S. (1974). Chain ordering in liquid crystals. II. Structure of bilayer membranes. Biochim. biophys. Ada 367, 165-76.

Phase transitions and fluidity characteristics 231 J. F. (1973). Theory of biomembrane phase transitions. J. chem. Phys. 58, 252-64. NAQVI, K. R., BEHR, J. P. & CHAPMAN, D. (1974). Methods for probing lateral diffusion of membrane components. Triplet-triplet annihilation and triplet-triplet energy transfer. Chem. Phys. Letters 26, 440-44. NICHOLLS, P. & MILLER, N. (1974). Chloride diffusion from liposomes. Biochitn. biophys. Acta 356, 184-98. NORDSIECK, H., ROSEVEAR, F. B. & FERGUSON, R. H. (1948). X-ray study of the stepwise melting of anhydrous sodium palmitate. J. chem. Phys. 16, 175-80. OHKI, S. (1969). The electrical capacitance of phospholipid membranes. Biophys. J. 9, 1195-205. OLDFIELD, E. & CHAPMAN, D. (1972). Dynamics of lipids in membranes: heterogeneity and the role of cholesterol. FEBS Lett. 23(3), 285-97. OLDFIELD, E., CHAPMAN, D. & DERBYSHIRE, W. (1971). Deuteron resonance; a novel approach to the study of hydrocarbon chain mobility in membrane systems. FEBS Lett. 16(2), 102-4. OLDFIELD, E., CHAPMAN, D. & DERBYSHIRE, W. (1972a). Lipid mobility in Acholeplasma membranes using deuteron magnetic resonance. Chem. Phys. Lipids 9, 69-81. OLDFIELD, E., KEOUGH, K. M. & CHAPMAN, D. (19726). The study of hydrocarbon chain mobility in membrane systems using spin-label probes. FEBS Lett. 20(3), 344-6. OVERATH, P., SCHAIRER, H. U. & STOFFEL, W. (1970). Correlation of in vivo and in vitro phase transitions of membrane lipids in Escherichia coli. Proc. natn. Acad. Sci. U.S.A. 67, 606-12. OVERATH, P. & TRAUBLE, H. (1973). Phase transitions in cells, membranes and lipids of Escherichia coli. Detection by fluorescent probes, light scattering and dilatometry. Biochemistry, N. Y. 12, 2625-34. PACHE, W. & CHAPMAN, D. (1972). Interaction of antibiotics with membranes. Chlorothricin. Biochitn. biophys. Acta 255, 348-57. PACHE, W-, CHAPMAN, D. & HILLABY, R. (1972). Interaction of antibiotics with membranes. Polymixin B and gramicidin S. Biochim. biophys. Acta 255. 3S8-64. PAGANO, R. E., CHERRY, R. J. & CHAPMAN, D. (1973). Phase transitions and heterogeneity in lipid bilayers. Science, N.Y. 181, 557-9. PALMER, K. J. & SCHMITT, F. O. (1941). X-ray diffraction studies of lipid emulsions. J. cell. comp. Physiol. 17, 385-94. PAPAHADJOPOULOS, D. (1968). Surface properties of acidic phospholipids: Interactions of monolayers and hydrated liquid crystals with uni- and bivalent metal ions. Biochim. biophys. Acta 163, 240-54. PAPAHADJOPOULOS, D., JACOBSON, K., NIR, S. & ISAC, T. (1973). Phase transitions in phospholipid vesicles. Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol. Biochim. biophys. Acta 311, 330-48. NAGLE,

PAPAHADJOPOULOS, D., POSTE, G., SCHAEFFER, B. E. & VAIL, W. J. (1974).

232

D. CHAPMAN

Membrane fusion and molecular segregation in phospholipid vesicles. Biochim. biophys. Acta 352, 10-28. PHILLIPS, M. C. & CHAPMAN, D. (1968). Monolayer characteristics of saturated 1,2-diacylphosphatidylcholine (lecithins) and phosphatidylethanolamines at the air-water interface. Biochim. biophys. Acta 163, 301-13. PHILLIPS, M. C. & FINER, E. G. (1974). The stoichiometry and dynamics of lecithin-cholesterol clusters in bilayer membranes. Biochim. biophys. Ada 356, 199-206. PHILLIPS, M. C , LADBROOKE, B. D. & CHAPMAN, D. (1970). Molecular interactions in mixed lecithin systems. Biochim. biophys. Acta 193, 35-44. PHILLIPS, M. C , WILLIAMS, R. M. & CHAPMAN, D. (1969). On the nature of hydrocarbon chain motions in lipid liquid crystals. Chem. Phys. Lipids 3, 234-44. PIERCE, W. C. (1935). Scattering of X-rays by polyatomic liquids, n-heptane. J. chem. Phys. 3, 252-5. RAISON, J. K., LYONS, J. M. & THOMSON, W. W. (1971). The influence of membranes on the temperature-induced changes in the kinetics of some respiratory enzymes of mitochondria. Archs. Biochem. Biophys. 142, 83-90. RAND, P., CHAPMAN, D. & LARRSON, K. (1974). Unpublished studies. RAND, P. & LUZZATI, V. (1968). X-ray diffraction study in water of lipids extracted from human erythrocytes. Biophys. J. 8, 125-37. REISS-HUSSON, F. (1967). Structure des phases liquide-crystallines de differents phospholipides, monoglyc6rides, sphingolipides anhydres ou en pr6sence d'eau. J. molec. Biol. 25, 363-82. ROJAS, E. & TOBIAS, J. M. (1965). Membrane model: Association of inorganic cations with phospholipid monolayers. Biochim. biophys. Acta 94 394404. ROTHMAN, J. (1973). The molecular basis of mesomorphic phase transitions in phospholipid systems. J. theor. Biol. 38, 1-16. ROTHMAN, J. E. & ENGELMAN, D. M. (1972). Molecular mechanism for the interaction of phospholipid with cholesterol. Nature New Biol. 237, 424SACKMANN, E. & TRAUBLE, H. (1972). Studies of the crystalline-liquid crystalline phase transition of lipid model membranes. I. Use of spin labels and optical probes as indicators of the phase transition. J. Am. chem. Soc. 94. 4482-97SALSBURY, N. J. & CHAPMAN, D. (1968). Physical studies of phospholipids. VIII. Nuclear magnetic resonance studies of diacyl-L-phosphatidylcholines (lecithins). Biochim. biophys. Acta 163, 314-24. SALSBURY, N. J., DARKE, A. & CHAPMAN, D. (1972). Deuteron magnetic resonance studies of water associated with phospholipids. Chem. Phys. Lipids 8, 142-51. SCHMITT, F. 0., BEAR, R. S. & CLARK, G. L. (1935). X-ray diffraction studies on nerve membrane. Radiology 25, 131-51. SCHREIER-MUCCILLO, S., MARSH, D., DUGAS, H., SCHNEIDER, H. & SMITH,


233

I. C. P. (1973). A spin probe study of the influence of cholesterol on the motion and orientation of phospholipids in oriented multi-bilayers and vesicles. Chem. Phys. Lipids 10, 11-17. SEELIG, J. (1970). Spin-lable studies of oriented smectic liquid crystals. (A model system for bilayer membranes.) J. Am. chem. Soc. 92, 3881-7. SEELIG, J. (1971). On the flexibility of hydrocarbon chains in lipid bilayers. J. Am. chem. Soc. 93, 5017-22. SEELIG, J. & NIEDERBERGER, W. (1974). Deuterium-labeled lipids as structural probes in liquid crystalline bilayers. A deuterium magnetic resonance study. J. Am. chem. Soc. 96, 2069-72. SEELIG, J. & SEELIG, A. (1974). Deuterium magnetic resonance studies of

phospholipid bilayers. Biochem. biophys. Res. Commun. 57, 406-7. A. M. & GERSHFELD, N. L. (i960). Interactions of veratrum alkaloids, procaine and calcium with monolayers of stearic acid and their implications for pharmacological action. J.gen.Physiol. 44, 345-63. SHEETZ, M. P. & CHAN, S. I. (1972). Effect of sonication on the structure of lecithin bilayers. Biochemistry, N.Y. n , 4576-81. SHANES,

SHEMYAKIN, M. M., OVCHINNIKOV, YU. A., IVANOV, V. T., ANTONOV, V. K., VINOGRADOVA, E. I., SHKROB, A. M., MALENKOV, G. G., EVSTRATOV, A. V., LAINE, I. A., MELNIK, E. I. & RYADOVA, I. D. (i960). Cyclo-

depsipeptides as chemical tools for studying ionic transport through membranes. J. Membrane Biol. 1, 402-30. SHIMSHICK, E. J. & MCCONNELL, H. M. (1973 a). Lateral phase separation in phospholipid membranes. Biochemistry, N.Y. 12, 2351-60. SHIMSHICK, E. J. & MCCONNELL, H. M. (19736). Lateral phase separation in binary mixtures of cholesterol and phospholipids. Biochem. biophys. Res. Commun. 53, 446-8. SHIPLEY, G. G. (1973). Recent X-ray diffraction studies of biological membranes and membrane components. In Biological Membranes (ed. D. Chapman & D. F. H. Wallach), pp. 1-85. New York: Academic Press. SMALL, D. M. (1967). Phase equilibrium and structure of dry and hydrated egg lecithin. J. Lipid Res. 8, 551-7. SPETH, V. & WUNDERLICH, F. (1973). A direct visualization of reversible transitions in biomembrane structure induced by temperature. Biochim. biophys. Ada 291, 621-8. STEIM, J., TOURTELLOTTE, M. E., REINERT, J. C , MCELHANEY, R. D. & RADER, R. L. (1969). Calorimetric evidence for the liquid-crystalline

state of lipids in a biomembrane. Proc. natn. Acad. Sci. U.S.A. 63, 104-9. STEWART, G. T. (1961). Mesomorphic forms of liquid in the structure of normal atheromatous tissues. J. Path. Bad. 81, 385-92. STIER, A. & SACKMANN, E. (1973). Spin labels as enzyme substrates. Heterogeneous lipid distribution in liver microsomal membranes. Biochim. biophys. Ada 311, 400-8. TARDIEU, A., LUZZATI, V. & REMAN, F. C. (1973). Structure and poly-

234

D

- CHAPMAN

morphism of the hydrocarbon chains of lipids: A study of lecithin-water phases. J. molec. Biol. 75, 711-33. TRAUBLE, H. (1971). Phasenumwandlungen in lipidenmogliche Schaltprozesse in biologischen Membranen. Naturwissenschaften 58, 277-84. TRAUBLE, H. (1972). Phase transitions in lipids. In Biomenibranes, vol. 3 (ed. F. Kreutzer & J. F. G. Siegers), pp. 197-227. New York: Plenum Press. TRAUBLE, H. & EIBL, H. (1974). Electrostatic effects on lipid phase transitions: Membrane structure and ionic environment. Proc. natn. Acad. Set. U.S.A. 71, 214-19. TRAUBLE, H. & HAYNES, D. H. (1971). The volume change in lipid bilayer lamellae at the crystalline-liquid crystalline phase transition. Chem. Phys. Lipids 7, 324-35. TRAUBLE, H. & SACKMANN, E. (1972). Studies of the crystalline-liquid crystalline phase transition of lipid model membranes. III. Structure of a steroid-lecithin system below and above the lipid phase transition. J. Am. chem. Soc. 94, 4499-510. TRUDELL, J. R., HUBBELL, W. L. & COHEN, E. N. (1973). The effect of two inhalation anaesthetics on the order of spin-labelled phospholipids. Biochim. biophys. Ada 291, 321-7. URRY, D. W. (1971). The gramicidin A transmembrane channel: A proposed n (L,D) he'ix- Proc. natn. Acad. Sci. U.S.A. 68, 672-6. VANDENHEUVEL, F. A. (1963). Study of biological structure at the molecular level with stereo model projections. I. The lipids in the myelin sheath of nerve. J. Am. Oil chem. Soc. 40, 455-71. VANDERKOOI, J. M. & CHANCE, B. (1972). Temperature sensitivity of fluorescence probes in the presence of model membranes of mitochondria.

FEBS Lett. 22, 23-6. Z., SALSBURY, N. J. & CHAPMAN, D. (1969). Physical studies of phospholipids. XII. Nuclear magnetic resonance studies of molecular motion in some pure lecithin-water systems. Biochim. biophys. Ada 183, 434-46.

VEKSLI,

VERKLEIJ, A. J., DE KRUYFF, B., VERVERGAERT, P. H. J. T H . , TOCANNE, J. F. & VAN DEENEN, L. L. M. (1974). The influence of pH, Ca2+ and protein

on the thermotropic behaviour of negatively charged phosphatidylglycerol. Biochim. biophys. Ada 339, 432-7. VERKLEIJ, A. J., VERVERGAERT, P. H. J. T H . , VAN DEENEN, L. L. M. & ELBERS,

P. F. (1972). Phase transitions of phospholipid bilayers and membranes of Acholeplasma laidlawii B visualized by freeze-fracturing microscopy. Biochim. biophys. Ada 288, 326-32. VOLD, M. J. (1941). Liquid crystalline, waxy and crystalline phases in binary mixtures of pure anhydrous soaps. J. Am. chem. Soc. 63, 160-8. VOLD, M., MACOMBER, M. & VOLD, R. D. (1941). Stable phases occurring between true crystal and true liquid for single pure anhydrous soaps. J. Am. chem. Soc. 63, 168-75. WARREN, B. E. (1933)- X-ray diffraction in long chain liquids. Phys. Rev. 44.


235

S. G. & CHAPMAN, D. (1965). Monte Carlo study of rotational premolting in crystals of long chain paraffins. Trans. Faraday Soc. 61, 2656-60. WHITTINGTON, S. G. & CHAPMAN, D. (1966). The effect of density on the configurational properties of long-chain molecules using a Monte Carlo method. Trans. Faraday Soc. 62, 3319-24. WILKINS, M. H. F., BLAUROCK, A. E. & ENGELMAN, D. M. (1971). Bilayer structure in membranes. Nature, Lond. 230, 72-6. WILLIAMS, R. M. & CHAPMAN, D. (1970). Phospholipids, liquid crystals and cell membranes. In Progress in the Chemistry of Fats and Other Lipids (ed. R. T. Holman), pp. 1-79. New York: Pergamon Press. ZACHARIASEN, W. H. (1935). The liquid 'structure' of methyl alcohol. J. chem. Phys. 3, 158-61.

WHITTINGTON,

Phase transitions in planar bilayer membranes.

Decreased fluidity of red cell membrane lipids in abetalipoproteinemia.

Effect of superparamagnetic iron oxide nanoparticles on fluidity and phase transition of phosphatidylcholine liposomal membranes.

Phase transitions in phospholipid model membranes of different curvature.

Lipids of rabies virus and BHK-21 cell membranes.

Investigation of phase transitions of lipids and lipid mixtures by sensitivity differential scanning calorimetry.

Differences in lipid fluidity among isolated plasma membranes of normal and leukemic lympocytes and membranes exfoliated from their cell surface.

Effects of halothane, thiopental, and lidocaine on fluidity of synaptic plasma membranes and artificial phospholipid membranes.

Phase transitions and phase separations in phospholipid membranes induced by changes in temperature, pH, and concentration of bivalent cations.

The effects of cell proliferation on the lipid composition and fluidity of hepatocyte plasma membranes.

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Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli.

Fluorine-19 nuclear magnetic resonance studies of lipid phase transitions in model and biological membranes.

The influence of phase transitions on the sorption of benzocaine by polyamide membranes [proceedings].

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Epigenetics and locust life phase transitions.

Quantum coherence and quantum phase transitions.

Formamidinium iodide: crystal structure and phase transitions.

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

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