The effect of salinity on the phase behaviour of purified phosphatidylethanolamine and phosphatidylglycerol isolated from a moderately halophilic eubacterium

Chemistry and Physics of Lipids, 56 (1990) 135--147 Elsevier Scientific Publishers Ireland Ltd.

135

The effect of salinity on the phase behaviour of purified phosphatidylethanolamine and phosphatidylglycerol isolated from a moderately halophilic eubacterium G.C. Sutton a,b, N.J. R u s s e l l a a n d P . J . Q u i n n b •Department of Biochemistry, University of Wales, P.O. Box 903, Cardiff CFI IST (U.K.) and bBiochemistry Section, Division of Biomolecular Sciences, King's College London, Campden Hill Road, London W8 7AH (U.K.) (Received May 30th, 1990; revision received August 20th, 1990; accepted August 22nd, 1990)

The phase behaviour of purified phosphatidylethanolamine and phosphatidylglycerol isolated from the moderately-halophilic eubacterium Vibrio costicola grown in 1 M or 3 M NaCl-containing medium has been studied as a function of NaCI concentration using differential-scanning calorimetry, freeze-fracture electron microscopy and X-ray diffraction. The two phospholipids exhibited complex phase behaviour which was dependent on the salinity of both the bacterial culture medium and the phospholipid resuspending solution. The phosphatidylethanolamine from cultures grown in 1 M or 3 M NaCl-containing media displayed a hexagonal-ll phase and this phase persisted at temperatures up to 20 degrees lower when the lipid was dispersed in 3 M compared with 1 M NaCI. The L° phase of phosphatidylethanolamine isolated from 1 M NaCI grown cultures was favoured less when the lipid was resuspended in 3 M compared with 1 M NaCI. The Lo "-" Lc phase transition temperature of the phosphatidylethanolamines coincided with ice formation. The phosphatidylglycerol samples did not exhibit non-lamellar phases over the temperature range + 50°C to - 50°C. An increase in salinity of the resuspending solution increased the L ~- L~ and Lp "* L c phase transition temperatures, indicating that the L~ and L c phases were both stabilised by the higher NaCI concentration. The L° ,-- L~ and Lo 4" L phase transition temperatures of phosphatidylglycerol isolated from 3 M NaCI cultures were higher than those from 1 M NaCI cultures.

Keywords: phosphatidylethanolamine; phosphatidylglycerol; sodium chloride; halophilic bacteria; hexagonal-II; lamellar.

Introduction Most biological membranes contain one or more classes of lipid which, in isolation and under physiological conditions, prefer a nonbilayer (e.g. hexagonal-II) phase [1,2]. Although a significant proportion of the total membrane lipids of many biological membranes may form non-bilayer structures in isolation, a bilayer arrangement dominates the structure of the native membranes, at least under conditions of biological relevance [3,4]. A knowledge of the biological function of non-bilayer forming lipids Correspondence to: N.J. Russell, Department of Biochemistry, University of Wales, P.O. Box 903, Cardiff CF1 1ST, U.K.

and the factors which constrain them in the bilayer configuration in biological membranes is fundamental to our understanding of membrane structure and stability [5,6]. The membranes of many Gram-negative bacteria contain a large proportion of unsaturated phosphatidylethanolamine, a non-bilayer-forming lipid, with lesser amounts of bilayer-stabilising lipids such as phosphatidylg.lycerol [7]. Phosphatidylethanolamines and phosphatidylglycerols both undergo a lamellar gel (L~) to lameUar liquid-crystalline ( L ) phase transition; phosphatidylethanolamines, in addition, undergo a L --- hexagonal-II transition at higher temperatures [8,9]. The precise temperatures of these transitions for both phospholipid classes depend upon a variety of factors, including the nature

0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

136

of the acyl chains and the dispersing solution [8 --14]. For example, an increase in the fatty acid chain length or an increase in the NaCl concentration of the dispersing solution result in an increasein the temperature of the L -~ Lp transition (TL) of both phosphatidylethanolamines and phosphatidylglycerols and a decrease in the temperature of the L -~ hexagonal-II transition (T N) of phosphatidylethanolamines; the magnitude of the salinity effect is usually greater on T n than on T L. However, other workers generally have not used NaCl concentrations high enough (i.e. > 1 M) to be relevant to the growth conditions of haiophilic organisms. In the moderately-halophilic eubacterium, Vibrio costicola, phosphatidylethanolamine and phosphatidylglycerol comprise > 80070 of the total membrane lipid and the ratio of these phospholipids and their fatty acid compositions depend on the salinity of the growth medium [15,16]. In response to an increase in the externai salinity, V. costicola, in common with many other Gram-negative halophilic and halotolerant eubacteria [17,18], increases the ratio of phosphatidylglycerol to phosphatidylethanolamine from < 0.5:1 in 1 M NaCI to > 1:1 in 3 M NaCl-containing media [15]. The simplest interpretation of these changes is that they are necessary to preserve the integrity of the lipid bilayer in the face of altered external salinity [17]. The fatty acyl residues of phosphatidylethanolamines and phosphatidylglycerols from V. costicola respond independently to changes in the salinity of the growth medium [16]. The major fatty acids of both phosphatidylethanolamine and phosphatidylglycerol from V. costicola grown in 1 M or 3 M NaCl-containing medium are 16:1c9 (c = cis), 16:0 and 18:1ci1; in general the sn-1 position of the glycerol contains a saturated and the sn-2 position an unsaturated fatty acyl residue. The isomers 16:1cll and 18:1c13, which are present in minor amounts (i.e. < 3.0 mo1070) in the phospholipids extracted from 3 M NaC1 cultures, are absent from the phospholipids of 1 M NaCI cultures. The fatty acids of phosphatidylethanolamine from cultures grown in 1 M and 3 M NaCI have the same unsaturation index and average chain

length. The main difference in the fatty acid composition of the phosphatidylethanolamines is a decrease in 18:1cll from 19.2 mo1070 in 1 M NaCI cultures to 14.9 mo1070 in 3 M NaCI cultures. In comparison with phosphatidylethanolamine, there are larger salinitydependent changes in the fatty acid composition of phosphatidylglycerol. The phosphatidylglycerol isolated from 1 M NaCI cultures is more saturated and has a larger C18/C16 ratio than the phosphatidylglycerol from 3 M NaCI cultures; an increase in 16:1c9, from 30.7 mo1070 in 1 M NaCI cultures to 38.5 mo1070 in 3 M NaC1 cultures, is associated with a corresponding decrease in 18:1cll content. Such changes in fatty acid composition are expected to affect the phase behaviour of both phosphatidylethanolamine and phosphatidyiglycerol. To gain an insight into the structural consequences and molecular basis underlying changes in lipid composition we examined the effect of NaCl upon the phase behaviour of purified phosphatidyiethanolamine and phosphatidylglycerol, isolated from V. costicola grown in 1 M and 3 M NaCI. Thermal, electron microscopic and dynamic X-ray diffraction studies have been combined to give a comprehensive thermodynamic description of the behaviour of these phospholipids in response to NaCl concentration.

Materials and Methods

Vibrio costicola (NRC 37001) was grown aerobically at 30°C in a complex liquid medium containing 0.3070 (w/v) Proteose Peptone (Difco), 0.3070 (w/v) Bacto Tryptone (Difco) and the appropriate concentration of NaC1 (AnalaR grade) as 10 l cultures in a Microferm fermentor (New Brunswick). Bacteria were harvested in the late-exponentiai growth phase using a Sharples continuous-flow centrifuge operated at 25,000 rev./min. Total lipid was extracted using the method of Bligh and Dyer as described by Kates [19]. Nonlipid contaminants were removed from the total-

137 lipid extract by liquid/liquid partition chromatography using Sephadex G-25 (fine grade, Pharmacia) as described by Wells and Dittmer [20]. The purified total-lipid extract was fractionated into its component phospholipids by carboxymethyl cellulose chromatography (CM 52 sodium form, Whatman) [21]. Phospholipid samples were shown to be > 98070 pure on the basis of two-dimensional thin-layer chromatographic separations and quantitation by phosphorus and fatty acid analyses [22]. Lipid samples were prepared from stock solutions in chloroform; before use the solvent was removed using a stream of dry nitrogen gas, the sample placed under vacuum for 16 h and hydrated with 80 vol.% of the appropriate NaC1 solution (pH 7.0). Differential scanning calorimetry was performed using a Perkin Elmer DSC-2 calorimeter at a sensitivity setting of 8.4 J s-I and a scanning rate of 10 centigrade degrees min-L This scan rate was chosen to be the same as that used in the X-ray diffraction experiments (see below) so as to facilitate direct comparison between the sets of data obtained using the different methodologies. Calorimetric enthalpies of samples were determined by cutting and weighing the areas under the transition peaks, which were compared with indium standard enthalpies. Samples for freeze-fracture were thermally quenched from the desired temperature using a jet of liquid nitrogen. A Polaron freeze-fracture machine was used to fracture the specimens at - l l 5 ° C and platinum-carbon replicas of the fracture surface were prepared. The replicas were cleaned using a solvent consisting of CHCI3/MeOH (l:l, v/v) before examination using a Philips EM301 transmission electron microscope operated at 100 kV. X-ray diffraction studies were performed using a monochromatic focused X-ray beam at station 8.2 of the Daresbury Synchrotron Laboratories (Daresbury, England). A cylindricallybent single crystal of germanium [23] and a long float-glass mirror were used to select monochromatic X-rays and to focus the beam horizontally to provide a beam size of 0.3 × 3.0 mm with about l0 ~° photons s-~ at 2.0 GeV and 200 mA

of electron beam current with the Wiggler operating at 5.0 tesla. A flat-plate camera was used with a linear-wire detector constructed at Daresbury. The detector contained 512 channels each of 0.193 mm. The detector response was determined by recording the signal from a fixed source accumulated over 1 h. X-ray scattering data was acquired in 255 consecutive time frames with an acquisition time for each frame of 4 s and a dead time between frames of 50/~s. A scan rate of 10 centigrade degrees min-1 was used to limit the exposure of the samples to the X-ray beam while permitting a large temperature range to be studied. Data was stored on a VAX 11/785 computer and the experimental data sets corrected for detector response using the OTOKO program at the Daresbury Laboratories. Calibration of the spacings was obtained using teflon [24] and dipalmitoylphosphatidylcholine in the Lp phase [25] as standards. The configuration of the camera and detector was such that diffraction spacings < 0.35 nm were not detected. The resolution of the camera/detector configuration was 0.15 nm for a diffraction spacing of 5.0 nm and 0.01 nm for a diffraction spacing of 0.36 nm. Samples were mounted between thin mica sheets set 1 mm apart and placed in a vertically-mounted THM600 cryostage (Linkam Scientific Instruments Ltd, Tadworth, U.K.) abutting to an electrically-heated silver block. A flow of nitrogen gas at - 1 5 0 ° C was passed internally through the sample holder. A TMSg0 control system fitted with a remote control unit (Linkam) provided the appropriate amount of power to the heating block to maintain the sample at the desired temperature. Temperature scans were performed by programming the control system to maintain the desired rate of heating or cooling between preset temperature limits. Two thermocouples placed adjacent to the sample in the sample holder were used to monitor the temperature internally. The analogue signals from these were recorded digitally, together with the data file, on the VAX 11/785 computer. Samples recovered from the apparatus were subjected to thin-layer chromatography and showed no signs of degradation.

138

Results

Assignment o f mesophases To compare the phase behaviour of the major phospholipids of V. costicola, the phosphatidyle t h a n o l a m i n e and p h o s p h a t i d y l g l y c e r o l extracted from cultures grown in 1 M or 3 M NaCl-containing media and dispersed in 1 M and 3 M NaCl were examined by differential scanning calorimetry, freeze-fracture electron microscopy and dynamic X-ray diffraction. The X-ray diffraction patterns which are representative of the four phases found in the phosphatidylethanolamine samples studied are shown in Fig. 1. A lamellar (L) arrangement was characterised by small-angle Bragg diffraction spacings which were in the ratio 1:1/2:1/3:1/4 etc. [26] (Figs. 1, a,b and c). The lamellar phases were subdivided according to the wide-angle data. A Lc phase was assigned where there were two or three diffraction peaks between 0.42 nm and 0.36 nm (Fig. la) and this mesophase was con-

firmed by the close-packed lameUar sheets observed in electron micrographs of freeze-fracture replicas (Fig. 2a). Hexagonal packing of the acyl chains in the Lo phase gave rise to only one wide-angle diffraction peak (Fig. lb) and a diffuse scattering profile was consistent with the disordered nature of the acyl chains which typifies the L phase (Fig. lc). Small-angle diffraction peaks in the ratio l:l/x/-3:l/x/-4:l/x/7:l/x/-9 (Fig. l d) are typical of a non-bilayer arrangement of lipids and can describe hexagonal or cubic mesophases [27]. Freeze-fracture electron micrographs of this phase (Fig. 2b) showed structures consistent with a hexagonal structure rather than a cubic arrangement. Since these studies were with diacylphosphatidylethanolamines, a hexagonal-II phase, rather than a hexagonal-I phase, was assigned. The same procedure was used to assign phases in the phosphatidylglycerol samples studied (see below).

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The thermal behaviour of the NaCI solutions was characterised separately from the lipid studies and the data is shown in Fig. 3 as dashed lines. Considerable supercooling was observed at a cooling rate of 10 centigrade degrees min-~ when compared to the cited values for the freezing of NaCI solutions [28]. When 1 M NaCl was cooled at 10 centigrade degrees min -~, it started to freeze with an exotherm between - 13 °C and - 1 5 °C followed by a larger exotherm between - 1 9 ° C and - 2 3 ° C (Fig. 3a, dashed lines). Melting commenced with a small endotherm between - 1 6 ° C and - 1 4 ° C followed by a larger endotherm between - 9°C and - 2 °C. By comparison 3 M NaCl froze at - 3 3 ° C and melting commenced with a large endotherm between - 2 6 ° C and - 2 0 ° C followed by a smaller one between - 1 8 ° C and - 1 4 ° C (Fig. 3b, dashed lines). Hence, 3 M NaCl remained liquid at temperatures 20°C lower than 1 M NaC1 and melted at temperatures 10°C lower than 1 M NaCl.

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139

Fig. 2. Electron micrographs of freeze-fracture replicas prepared from phosphatidylethanolamine isolated from V. costicola grown in (a) 1 M NaCl-containing medium and dispersed in 1 M NaCI and Co) 3 M NaCl-contalning medium and dispersed in 80% by vol. 3 M NaC1 (pH 7.0). Samples were thermally quenched from (a) -25°(2 or (b) + 30°C. Bars represent 100 rim.

ethanolamine extracted from cells grown in 1 M NaCI (i.e. "1 M phosphatidylethanolamine") which had been dispersed in 1 M NaC1 is shown in Fig. 4a. On cooling, this sample exhibited a hexagonal-I1 phase from + 5 0 ° C to + 18°C. The presence of a mixture o f hexagonal-II and Lo phases between + 25 °C and + 18 °C was confirmed by electron micrographs o f freeze-fracture replicas thermally quenched from 20°C and formation o f this mixture o f phases was associated with an exothermic transition having an enthalpy of 0.54 kJ tool -1 (Fig. 3a). The L phase had a lamellar repeat o f 5.6 nm. A L~ phase with a lamellar repeat of 6.1 nm was observed over the temperature range + 2°C to - 1 4 ° C . The onset o f the L~ phase was associated with an exothermic transition of enthalpy 0.27 kJ mol-L The L~ ~ L phase transition at - 1 4 ° C coincided with ice formation in the sample. On subsequently heating the frozen sample, the sequence o f transitions observed during cooling was reversed; the L~ ~ L~ transition at - 8 ° C and the end of the L~ phase at + 4°C showed some hysteresis, while the hexag-

onal-II phase was present at a lower temperature ( + 6°C) during heating compared with cooling. The appearance o f the hexagonal-II phase at t h e expense of the L phase was associated with an endotherm of enthalpy 0.75 kJ mol -~ (Fig. 3a). Upon cooling, the L~ ~ L c phase transition occurred at - 14°C with the 1 M NaC1 solution freezing at - 1 3 ° C . Similarly, upon heating the 1 M NaCI solution melted at - 9 ° C with the L c ~ Lp phase transition taking place at - 8 °C. When the 1 M phosphatidylethanolamine was dispersed in 3 M NaCI (Fig. 4b), the sequence o f phases during cooling was the same as for the lipid dispersed in 1 M NaCI, except that the corresponding phase-transition temperatures in the higher salt concentration were approximately 20 centigrade degrees lower. The hexagonal-II phase was observed down to - 5 ° C and the L~ ~ Lc transition took place at - 28 °C compared with values of + 18°C and - 14°C, respectively, for the lipid dispersed in 1 M NaCI. An endotherm having an enthalpy value o f 5.79 kJ mol "l, which spanned a temperature range o f 45 degrees between + 20°C and - 2 5 ° C , was asso-

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142 ciated with the transition from the hexagonal-II to the lamellar phases and the L~ --- L transition occurred as the sample started to freeze (Fig. 3b). The large temperature range over which the hexagonal-II to lamellar transition took place indicates that this is not a cooperative transition. Upon subsequently reheating the sample dispersed in 3 M NaCI, pronounced hysteresis was observed (> 20 degrees) especially in the L~--- L~ transition and the temperature of the final disappearance of the L~ phase. The transition from lamellar to hexagonal-II phase was associated with an endotherm of enthalpy 3.57 kJ mo1-1 which showed non-cooperative behaviour and spanned a temperature range of 35 °C (Fig.

3b). The corresponding phase behaviour of phosphatidylethanolamine extracted from cells grown in 3 M NaCl-containing medium (i.e. "3 M phosphatidylethanolamine") dispersed in 1 M or 3 M NaC1 is shown in Figs. 4c and 4d. On cooling the lipid dispersed in 1 M NaC1, the hexagonal-II phase persisted at temperatures down to - 9 ° C whereupon it converted directly to the L, phase; an L~ ~ ~ transition was observed at - 2 2 ° C . As with the 1 M phosphatidylethanolamine, the L phase was present only when the NaCI solution underwent freezing. On heating, the L c phase converted to L~ and hexagonal-II phases at - 10°C. The end of the L~ phase was associated with an endotherm having an enthalpy value of 1.10 kJ tool-1 (Fig. 3c). When the 3 M phosphatidylethanolamine was dispersed in 3 M NaCI, only hexagonal-II and L~ phases were observed; the two phases coexisted for > 20 centigrade degrees during both cooling and heating, which again indicated that the transition was noncooperative (Fig. 4d). The onset of the hexagonalII phase at - 12°C and the final disappearance of the L~ phase at + l l°C were associated with exotherms having enthalpy values of 0.79 and 3.95 kJ mol-~, respectively (Fig. 3d).

Phase behaviour of phosphatidylglycerols The thermograms and phase behaviour for phosphatidylglycerol isolated from V. costicola grown in media containing either 1 M or 3 M NaC1 are shown in Figs. 5 and 6, respectively.

Unlike the phosphatidylethanolamines, no hexagonal-II phase was observed in any of the phosphatidylglycerol samples and the lamellar phases showed few higher orders in the smallangle region of the X-ray diffraction patterns. Using differential-scanning and X-ray diffraction no phase transitions were observed in 1 M phosphatidylglycerol dispersed in 1 M NaCI, the L a phase being present over the whole temperature range studied during both heating and cooling (Figs. 5a and 6a). The lamellar repeat distance of the Lo phase increased from 5.2 nm at 44°C to 5.7 nm at - 5 0 ° C . When 1 M phosphatidylglycerol dispersed in 3 M NaCI was cooled, there was a Lo --" L~ phase transition at - 2 0 ° C , which corresponded to an exotherm having an enthalpy value of 1.85 kJ mol-t (Figs. 5b and 6b). As observed with the 1 M phosphatidylglycerol dispersed in 1 M NaCI, the lamellar repeat distance of the Lo phase increased slightly with decreasing temperature from 5.0 nm at 46°C to 5.4 nm at - 16°C. The transition from the L~ to the Lc phase at - 2 8 ° C occurred just before the sample froze at - 3 2 ° C . When 1 M phosphatidylglycerol dispersed in 3 M NaCI was heated, the L phase converted directly to the L phase at - 2 1 °C, a temperature that coincided with the ice-melting temperature (Fig. 5b). When 3 M phosphatidylglycerol was dispersed in 1 M NaCI, a transition from the L to the L~ phase was observed at - 2 4 ° C during cooling (Fig. 6c). This transition was reversible and showed hysteresis upon heating, when it occurred at - 7 ° C . These transitions corresponded to the freezing and melting of the sample as observed in Fig. 5c. When 3 M phosphatidylglycerol dispersed in 3 M NaCI was cooled, there was a transition from the Lo to the L~ phase between - 5 ° C and - 1 5 ° C and a L~ ~ Lc phase transition at - 37°C (Fig. 6d). The onset of the L ~ L~ transition was associated with an exotherm having an enthalpy value of 0.94 kJ mol-~ (Fig. 5d). The L~ --- L c phase transition coincided with the onset of freezing of the NaCI solution. On heating, the Lc ~ L~ phase transition occurred at - 2 0 ° C , which was when the NaCI solution melted; the L, --" Lo phase transition at - 3 °C was associated with an endotherm a

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3"0

3-0

t'0 0"45

1"0 0.45

0'35

0.35

~---e

~

0--0

H

(

Cool

L=

) (.-- LB,---)

H

<

Cool

L=

> ~ - ~ LB ~L~

,

,

+50

Heat

,

+30

<

,

,

+10

L=

,

,

=

i

=

,

=

-10

-30

> (

LB ... ~,

,

+50

-50 (°C)

Heat

,

i

+30

(

i

,

i

+10

L=

-10

,

i

L

-30

J

-50 (°C)

) Lee Lc-m~

Fig. 6. Diffraction spacings and phase assignments calculated from X-ray diffraction data for phosphatidylglycerols isolated from V. costicola grown in l M (a and b) or 3 M (c and d) NaCI-containing media and dispersed in 80% by vol. l M (a and c) or 3 M (b and d) NaCI (pH 7.0) during cooling and heating scans between + 50°C and - 50°C at l0 centigrade degrees rain-' (diffraction spacings for a given phase were identical during heating and cooling, therefore those for heating are not shown). Diffraction spacings are expressed as nm in order to facilitate direct comparison with the text and those belonging to a group have been connected by lines.

145 having an enthalpy value of 0.71 kJ mol -~ (Fig. 5d). Discussion

This study represents the first report of the effect of molar concentrations of NaCI on the phase behaviour of acyl phospholipids extracted from a halophilic eubacterium, i.e. concentrations of NaCl which are physiologically relevant to such organisms. We show that purified phosphatidylethanolamine and phosphatidylglycerol isolated from the moderately-halophilic eubacterium Vibrio costicola display complex phase behaviour, including the formation of nonbilayer lipid phases, which dePends on the NaCl concentration of both the bacterial culture medium and the phospholipid resuspending solution. An increase from 1 M to 3 M in the NaCl concentration of the dispersing solution of phosphatidylethanolamine isolated from cultures grown in 1 M NaC1 (i.e. "1 M phosphatidylethanolamine") decreases the temperature at which the L phase is formed and the temperatures of the hexagonal-II ~ lamellar and L# ~ Lc transitions, on cooling, by 17, 23 and 14 centigrade degrees, respectively. Thus, a high NaCl Concentration stabilises the hexagonal-II phase with respect to the lamellar phase and destabilises the L and Lc phases. The effect of NaCl in the dispersing solutions is twofold. Increasing the NaCl concentration increases the ionic strength of the solution whilst decreasing its melting and freezing points. Since the formation of the L phase is linked to the physical state of the NaC1 solution, the Lc phase only being formed after the solution has frozen; increasing the NaCl concentration will result in a lower L0 ~ L transition temperature. When the 1 M phosphatidylethanolamine dispersions are reheated, the Lc~Lp transition in 3 M NaCI occurs at a higher temperature than in 1 M NaC1 ( - 3 °C and - 8 °C, respectively). The transition temperature in 3 M NaCI occurs 15 centigrade degrees above the melting point of the NaCI solution, whereas in 1 M NaCI the L~ ~ Lo transition takes place only during melting of the NaC1 solution. Thus it can be con-

eluded that, whereas the formation of the Lc phase is triggered by the freezing of the solution, it is the ionic strength which determines the stability of the Lc phase upon reheating, with a high NaC1 concentration having a stabilising effect. A similar effect on the hexagonal-II to lamellar transition temperature to that seen with 1 M phosphatidylethanolamine is observed with 3 M phosphatidylethanolamine - - i.e. when the NaCI concentration is increased the hexagonal-II phase persists at lower temperatures. It is known that increasing the NaCI concentration of the dispersing solution increases the Lo ~ L# transition temperature, whilst decreasing the L ** hexagonal-II transition temperature in phosphatidylethanolamines [8,29]. It seems unlikely that this reduction in the hexagonal-II to lamellar phase transition temperature (Ta) is achieved by a simple electrostatic screening of the head-group dipoles, since Seddon et al. [8] showed that lipids which have similar dipolar characteristics do not have the same response to NaCI concentration. Thus it would appear that monovalent ions affect the interactions of water molecules with the head-group region of the phospholipids and may displace the water of hydration [10,30]. This would effectively dehydrate the headgroups, reducing their surface area and favouring hexagonal-II phase formation [31]. This effective dehydration of head-groups by NaC1 produces similar shifts in the values of T L and T H to those obtained when the hydration of the phospholipid head-groups is lowered by reducing the water content of the sample [8]. There are also large differences in the phase behaviour of 1 M and 3 M p hosphatidylethanolamines when they are dispersed in the same NaCI concentration. We presume that these differences in phase behaviour result from changes in the fatty acid composition of the phosphatidylethanolamine which occur when the growth NaCI concentration is altered [16]. The 3 M phosphatidylethanolamine, in contrast to the 1 M NaC1 phosphatidylethanolamine, does not exhibit an L phase when dispersed in 1 M or 3 M NaCI. Direct transitions from the lamellar-gel state (L#) to the hexagonal-II phase, without an intervening fluid-lamellar phase (Lo), have been

146 reported previously for phosphatidylethanolamines in NaCI dispersions [11,32]. It appears, therefore, that the fatty acid composition of the 3 M phosphatidylethanolamine results in the hexagonal-II phase being more stable than the L phase and consequently the hexagonal-II --~ L and L ~ Lp transitions coalesce. The temperature which marks the end of the hexagonal-II phase for 3 M phosphatidylethanolamine is up to 20 centigrade degrees lower than for l M phosphatidylethanolamine (cf. Figs. 4a with 4c, and Figs. 4b with 4d). The L phase was not observed in 3 M phosphatidylethanolamine dispersed in 3 M NaCl and the L~-*L~ transition temperature is 8 centigrade degrees lower for 3 M phosphatidylethanolamine than for l M phosphatidylethanolamine when they are dispersed in 1 M NaC1. Again we conclude that the apparently small changes in fatty acid composition produce significant differences in the observed phase behaviour. The influence of fatty acyl chain length, unsaturation and branched fatty acids on the phase transition temperatures of phosphatidylethanolamines has been reported [8 --11,33,34], but these studies have largely used diacylphosphatidylethanolamines containing a single fatty acid. The present study has used phospholipids of biological origin having a heterogeneous fatty acid composition, thus making it difficult to predict the qualitative effects underlying the observed phase behaviour. However, some general points can be noted. The major difference in the fatty acid compositions of l M and 3 M phosphatidylethanolamines lies in the position of the double bonds: 3 M phosphatidylethanolamine has 16:lcll and 18:lcl3 isomers which are not present in 1 M phosphatidylethanolamine. Thus, the overall double-bond position in 3 M phosphatidylethanolamine is further from the centre of the acyl chain compared with that in 1 M phosphatidylethanolamine. Using a series of mono-unsaturated phosphatidylcholines, Barton and Gunstone [35] showed that moving the double bond away from the central position increased the gel to liquid-crystalline phase transition temperature. Hence, it can be predicted that the changes in double-bond ~ position of phosphatidylethanolamine in V. costicola would increase the

temperature of the gel to liquid-crystalline phase transition. This prediction is consistent with the phase behaviour of 1 M and 3 M phosphatidylethanolamine observed experimentally. The phosphatidylglycerol samples exhibit no hexagonal-II phase over the temperature range studied, which is consistent with its role as a bilayer-stabilising phospholipid. Increasing the NaCI concentration in which the 1 M phosphatidylglycerol is dispersed results in L~ and L c phases which are not observed when the lipid is dispersed in 1 M NaCI. Similarly for 3 M phosphatidylglycerol, increasing the NaCI concentration of the dispersions increases the L ~ L~ and L0 ~ L transition temperatures. These observations are consistent with previous reports of an increase in the Lo ~-~L~ transition temperature with molar increases in NaCI concentration [12]. As was noted above for the phosphatidylethanolamine samples, it is difficult to make further worthwhile comparisons of the phase behaviour of the phosphatidylglyerol samples with published data because of the paucity of literature regarding the phase behaviour of phosphatidylglycerols; it is also difficult to relate studies on synthetic samples, which usually have simple fatty acyl compositions but may have a racemic glycerol, to biological samples which generally have more complex fatty acyl compositions but a stereospecific glycerol. There are especially few reports of the effects of NaCI concentration on the phase behaviour of phosphatidylglycerols and, moreover, studies have generally employed phospholipids containing a single saturated fatty acyl species rather than unsaturated and mixed fatty acyl chains which are more biologically relevant. As was observed with the phosphatidylethanolamines, the phase behaviour of phosphatidylglycerols isolated from V. costicola grown in media containing different NaCI concentrations but dispersed in the same salinity are dissimilar. This is a consequence of the changes in the fatty acid composition of the phosphatidylglycerols which occur when the salinity of the growth medium is altered [16]. The 3 M phosphatidylglycerol was more saturated, had a different double-bond isomeric distribution and a shorter average chain length compared with the

147

1 M phosphatidylglycerol. These changes result in a higher L. -* L0 transition temperature in the 3 M phosphatidylglycerol than the 1 M phosphatidylglycerol.

13

Acknowledgements

16

This work was funded by a research grant from the Agriculture and Food Research Council (to P.J.Q. and N.J.R.). We thank Dr A.P.R Brain for technical assistance in the preparation of the freeze-fracture replicas.

17

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