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Phase behavior of synthetic N-acylethanolamine phospholipids
('hemistliv and Ptu'sics ojLipids, 42 11986) 249 260
249
I lsc~ier Scientific Publishers Ireland ltd.
PHASE BEHAVIOR OF SYNTHETIC N-ACYLETHANOLAMINE PHOSPHOLIPIDS
.I.1. NEWMAN*, D.I.. SI[IRS**, W.tl. ANI)I'2RSON and tt.tl.O. S('ItMID +
The tlormel Institute, Unirersity oJ Minnesota, Austin, MN 55912 (U.S.A.) Received .luly 2 l~t. 1986 revision received October 23rd, 1986 accepled October 23rd. 1986 Both saturated and unsaturated N-acylethanotamine phospholipids form lamellar structures t*hen dispersed in buffer. The addition of excess ('a 2÷ (('a2*/N-acytphosphatidylethanolamine > 0.5) results in precipitation. Vreeze-fracture replicas indicate that the addition of ('a 2÷ to the unsaturated lipid results in a non-bilayer structure ,~hile the ('a2+-complex of the saturated lipid is lamellar. Since unsaturated phospllatidylethanolaminc (PE) is a non-bilayer lipid, its N-acylalion with a saturated fatty acid converts a non-bilayer lipid into an acidic bilayer lipid capable of interacting with ('a ~÷ to return to a non-bilayer structure. ( a 2÷ may lhereby exert an influence on membrane phenomena by regulating phase behavior ~ithin certain membrane domains. Differential scanning calorimetry (DSC) indicates that N-acylation ol unsaturated Pt( ,s, ith a saturated fatty acid also results in changes in thermotropic phase behavior. Therefore, N-acylalion may affect fluidity ~ithin certain membrane domains.
Keywordsu N-acylphosphatidylethanolamine; phosphatidylelhanolamine; differential scanning calorimetry: freeze-fracture electron microscopy.
Introduction N - A c y l e t h a n o l a m i n e p h o s p h o l i p i d s o c c u r in m i c r o o r g a n i s m s [ 1 , 2 ] , p l a n t s 5] a n d certain v e r t e b r a t e tissues [6 9]. In the slime m o l d Dictyostelium discoideum, t h e y are present at a level o f a b o u t 10% o f t o t a l p h o s p h o l i p i d in the developing a m o e b a e b u t disappear d u r i n g t h e aggregation stage o f d e v e l o p m e n t [2]. They also disappear f r o m the seeds o f higher plants u p o n g e r m i n a t i o n [5]. In m a m m a l i a n cells, N - a c y l e t h a n o l a m i n e p h o s p h o l i p i d s can a c c u m u l a t e as the result of degenerative change, s u c h as t h a t o b s e r v e d in the granular cells of the e p i d e r m i s [3
*Present address: Worldwide tnnochem. Research and Development. S.(. Johnson & Son, Inc., 1525 llowe Street, Racine, Wl 53403, U.S.A. **Present address: Oklahoma Medical Research toundation, 825 NE 13th Street, Oklahoma City, OK 73104, U.S.A. +To whom correspondence should be addressed, Abbreviations: DS(', differential scanning calorimetry; HII, Hexagonal II; PC. phosphatidylcholine; PI-, phosphatidylethanolamine; constituent fatty acids are identified by parenthesis, e.g,, (dioleoyl)P[,;; the terms ethanolamine phospholipids and N-aeylethanolamine phospholipids indicate the actual or possible presence of diacyl, alkylacyl and alkenylacyl species, 000%3084/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
250 [6,7], in degenerating baby hamster kidney cells [10], in canine myocardial infarct [11] and in post-decapitative cerebral ischemia of the immature rat [12]. In both anaerobic bacteria [1] and mammalian tissues [13 18], N-acylethanolamine phospholipids are formed by acyl transfer to the amino group of ethanolamine phospholipids. In mammalian cells, this transfer involves 1-O-acyl g, oups of PE [14], phosphatidylcholine (PC) [15], and cardiolipin [16], and is catalyzed by a membrane-bound enzyme which exhibits an alkaline pH optimum and requires high amounts of Ca 2÷ for activity [ 13 -18]. Conversion of ethanolamine phospholipids to their N-acyl derivatives results not only in the addition of a hydrophobic moiety but also in the formation of an acidic phospholipid due to the loss of the free amino group. In order to evaluate the potential physiological or pathological significance of N-acylation reactions involving membrane phospholipids, it is of interest to determine the physical properties and phase behavior of N-acylated glycerophospholipids. The presence of Ca 2÷ has been shown to have a dramatic effect on the structures adopted by dispersions of acidic phospholipids and by mixed lipid systems containing them. Ca 2÷ complexes of some of these lipids, such as cardiolipin and phosphatidic acid, adopt hexagonal 11 (HII) phases [19,20], while others adopt various lamellar structures [21,22]. In liposomes composed of a mixture of acidic phospholipids with non-bilayer (HII) unsaturated PE, in which the bilayer structure is stable in the absence of Ca 2÷, the addition of Ca 2÷ triggers a bilayer to Hu transition [23,24]. The exact mechanism by which this occurs probably depends on the nature of the acidic phospholipid present [25]. Non-bilayer structures referred to as lipidic particles have been observed in these systems as probable intermediate structures in this process [26]. There is evidence for the existence of such structures within intact biological membranes as well. These lipidic particles, which have only been found in systems containing HII lipids, have been postulated to play a role in membrane fusion events, such as endocytosis and exocytosis, as well as in transbilayer transport phenomena [25 27]. Thus, the ability of Ca 2+ to modulate the occurrence of these lipidic particles may provide a means of regulation of certain membrane processes. Conversion of PE, a non-bilayer lipid, to N-acyl-PE may provide a mechanism for isothermal control over the occurrence of lipidic particles and thus over fusion of membrane transport processes. In this paper, we report the phase behavior of saturated and unsaturated N-acyl-PE using DSC and freeze-fracture electron microscopy.
Experimental N-Acyl-PE synthesis 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine was obtained from Calbiochem:
1,2-dipalmitoyl- and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine were obtained
251 from Avanti. Fatty acids were purchased from Sigma. 1,2-Dilauroyl-sn-glycero-3phospho(N-lauroyl)ethanolamine and 1,2-dipahnitoyl-or 1,2-dioleoyl-sn-glycero-3phospho(N-palmitoyl~ethanolamine were synthesized by acylation of (dilauroyl)PE, (dipalmitoyl)PE, and (dioleoyl)PE, respectively. The fatty acid (2 mmol) was reacted with N,N'-carbonyldiimidazole (1.8 mmol) in dry benzene for 4 h to form the fatty acid imidazolide. The PE (1 mmol) was then added to the reaction mixture which was stirred overnight. Thin-layer chromatography on silica gel H plates (chloroform/methanol/conc. ammonium hydroxide, 80:20:2, by vol.)showed that the reaction was essentially complete. The synthetic N-acyl-PE was purified by silicic acid column chromatography. The crude product was applied to the column in chloroform and chloroform or chloroform/methanol mixtures were used for elution. The pure N-acyl-PE was eluted with chloroform/methanol (90: 10, v/v). Thin-layer chromatography of a heavily-loaded sample revealed no detectable impurities. The migration rate of the purified product was identical to that of N-acyl-PE previously synthesized and characterized [9,11 18].
Preparation of lipid dispersions A chloroform solution of the lipid was evaporated to dryness under a stream of nitrogen. Residual solvent was removed by drying the lipid under vacuum overnight. The saturated phospholipids were then dispersed in 100 mM NaC1, 50 mM Tris HC1 (pH 7.2) by extensive vortexing at or above 55°C, whereas, the unsaturated lipid was vortexed at room temperature. The buffer was diluted to 20% glycerol in samples for freeze-fracture to prevent freeze damage or to 40% ehtylene glycol for calorimeter samples when scanning from sub-zero temperatures was desired. To obtain samples with the desired Ca2+/N-acyl-PE molar ratios, appropriate aliquots of a 100 mM or 200 mM CaCI 2 stock solution were added.
DSC DSC thermograms were obtained using a MicroCal MC-2 differential scanning calorimeter. The sample cell contained 1.22 ml of the lipid dispersion (2.5-7.5 ~mol/ml or 2 6 mg/ml), while the reference cell held the same volume of the buffer used in the dispersion. Heating scans were performed at a scanning rate of 10° or 20°C/h.
Freeze-fracture electron microscopy Dispersions or precipitates were transferred to specimen holders equilibrated at the desired temperatures and were then rapidly quenched in liquid freon or liquid propane at liquid nitrogen temperature. Replicas were prepared by standard freeze-fracture techniques with a Balzers BAF 300 apparatus. Replicas were floated off in water and cleaned with chloroform, when necessary. Micrographs of replicas were obtained with a J EOL 100-S electron microscope.
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Results and Discussion When vortexed at the appropriate temperatures, both the saturated and unsaturated N-acyl-PE formed milky dispersions. In contrast, (dioleoyl)PE, which is a non-bilayer lipid, would not form a stable homogeneous dispersion at room temperature. When excess Ca > (Ca>/N-acyI-PE > 0.5) was added to the concentrated dispersions of either the saturated or unsaturated N-acyl-PE used in freeze-fracture experiments, a precipitate formed, as is common with other acidic phospholipids. In the less concentrated calorimeter samples, the formation of a precipitate was not immediately apparent. Therefore, it appears that the effect of Ca =+ is dependent on the actual lipid or cation concentration as well as 1he Ca2+/N acyl-PE ratio.
Calorimetry DSC thermograms of N-lauroyl(dilauroyl)PE reveal a narrow energetic endotherm at 32°C indicative of a gel-to-liquid-crystal transition (Fig. 1 ). The transition half-width measured 0.3°C in this heating scan obtained at a heating rate of IO°('ih. This value is comparable to those obtained for saturated PE [28]. The transition temperature of the parent compound is 30.5°C, very close to that of the N-acylated derivative [28]. Tile gel-to-liquid-crystal transition of N-pahnitoyl(dipahnitoyl)Pl,~ also occurs at approximately the same temperature as that of the parent compound, (dipalmitoyl)PE (data not shown). The fact that N-acylation of saturated PE with the same fatty acid does not effect a change in the transition temperature indicates that the change in head group region of the lipid has a negligible effect on the transition temperature of the lipid. Figure 2 shows a series of thermograms obtained after addition of excess ('a 2+ to N-lauroyl(dilauroyl)PE. In the first scan (A), the original peak at about 32°( ' is present as well as an additional endotherm at 380( ". Successive heating scans (B and C) reveal an increase in the enthalpy of the 380( ` transition concomitant with a decrease in the enthalpy associated with the 32°( ` transition. In the third scan of the series (CL the lower-temperature transition is no longer present. Addi, tionally, two unresolved peaks can be seen at about 3~)° and 41°C. These resulis suggest that the Ca>-complex melts at a higher temperature than does the free lipid and is apparently formed at higher temperatures. This may be indicative of limited initial accessibility of the acidic lipid head groups to external Ca 2+. The heating scan for commercial (dioleoyl)PE (Avanti) indicates that the reversible gel-to-liquid-crystal transition occurs at 6°C (data not shown). The transition half-width of 0.8°C for this unsaturated lipid is greater than that obtained for saturated PE and N-acyl-PE. This may be due in part to the higher heating rate of 20°C/h required for scanning from sub-zero temperatures on the MC-2 instrument. While previous NMR studies have shown that a bilayer ---* ttll transition occurs between 7°C and 12°C in this PE [291, no endotherm other than
253
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Fig. 1. DSC scan of a dispersion of N-lauroyltdilauroyl)PE in 5 0 mM Tris-ttC1 buffer (pH 7.2). at a heating rate of 10°C/h.
the main transition is apparent in this scan. However, a relatively weak exotherm at 0°C can be seen when the sensitivity is increased. The detection of the bilayer HII transition using higher sensitivity DSC has recently been reported [30]. The enthalpy of this transition was dependent upon the length of time the lipid was incubated at 0°C. This phenomenon may be related to the exotherm observed in our study at 0°C. The synthetic N-palmitoyl derivative of (dioleoyl)PE exhibits its main endotherm at +2°C with a slight shoulder at about 0°C (Fig. 3). Therefore, N-acylation of (dioleoyl)PE with saturated palmitic acid results in an increase in the gel-toliquid-crystal transition by 8°C. Thus, it would appear that fluidity within membrane domains could potentially be affected by N-acylation of PE, depending on the nature of the acyl chain involved. As is the case with the saturated N-acyl-
254
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PE, the addition of excess Ca 2+ to this unsaturated N-acyl-PE results in an increase in the transition temperature to 6°C, as shown in the successive thermograms in Fig. 4. A minor exotherm at 0°C can also be seen in these scans.
Freeze-fracture Electron micrographs of freeze-fracture replicas of N-lauroyl(dilauroyl)PE dispersions at temperatures up to 65°C reveal liposomal structures (Fig. 5), while
255
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Fig. 3. DSC scan of a dispersion of N-palmitoyl(dioleoyl)PE in buffer plus 40% ethylene glycol at a heating rate of 20"C/h.
replicas of the Ca 2+ complex of this lipid indicate a stacked lamellar structure (Fig. 6). These results are similar to those obtained for saturated phosphatidylglycerol [31] and phosphatidytserine [32]. Quenching from temperatures above 30°C may not prevent a possible transition from an HII to a lamellar phase [26]. Thus, these freeze-fracture results are not sufficient to determine the correct phase of this lipid at 45°C. However, when dispersions of the lipid (in the absence of Ca 2+) were heated to temperatures in excess of 65°C, no precipitate indicative of the HII phase was observed, and 31P-NMR spectra indicated lamellar structure for this lipid and the Ca 2÷ complex up to 45°C. Replicas of N-palmitoyl(dioleoyl)PE dispersions at room temperature up to 55~C show a lamellar liposomal structure (Fig. 7) as opposed to the HII of (dioleoyl)PE [33]. Although freeze-fracture data cannot definitively identify lamellar structures above 30°C, these results indicate that this lipid remains in the bilayer form up to 30°C. Because the N-acylated derivative of a non-bilayer
256
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TEMPERATURE (oc) Fig. 4. Consecutive DSC scans o f a dispersion of N-palmitoyl(dioleoyl)PE in buffer plus 40% ethylene glycol with excess Ca 2+ (Ca2+/N-acyl-PE = 0.6) at a heating rate o f 20*C/h.
Fig. 5. Electron micrograph of a freeze-fracture replica of an N-lauroyl(dilauroyl)PE dispersion in buffer plus 20% glycerol at 45"C.
257
Fig. 6. Electron micrograph of a freeze-fracture replica of the Ca2+-complex of N-palmitoyl(dioleoyl)PE at 45°C, formed by the addition of excess Ca 2÷ (Ca2"/N-acyl-PE = 0.6) to a dispersion of the lipid in buffer plus 20% glycerol.
Fig. 7. Electron micrograph of a freeze-fracture replica of a dispersion of N-palrnitoyl(dioleoyl)PE in buffer plus 20% glycerol at 45"C.
258
Fig. 8. Electron micrograph of a freeze fracture replica of the Ca2÷-complex of N-palmitoyldioleoyl)PE at 55"C, formed by the addition of excess Ca2÷ (Ca2÷/N-acyl-PE= 0.6) to a dispersion of the lipid in buffer plus 20% glycerol.
lipid forms lamellar structures, N-acylation may have an overall bilayer-stabilizing effect in certain membrane domains. The structure of the Ca 2÷ complex of this unsaturated N-acyl-PE was also investigated by the freeze-fracture technique. As is the case with cardiolipin [34], Ca 2÷ appears to convert the saturated form of this acidic phospholipid to a HII phase (Fig. 8). Thus, although N-acylation converts the non-bilayer unsaturated PE into a bilayer lipid, the addition of Ca 2÷ can reverse the bilayer-stabilizing effect. These results suggest that N-acylation o f unsaturated PE can have a bilayerstabilizing effect within membrane domains. In addition, Ca 2÷ is capable of modulating this stabilizing effect. This could possibly affect the occurrence of nonbilayer structures, such as lipidic particles and could thereby affect membrane fusion and transport phenomena. Depending on the nature of the added acyl group, N-acylation could also have an effect on membrane fluidity in certain domains. Further studies of mixed lipid systems containing N-acyl-PE, PE and other lipids, should reveal more about the potential effects of N-acylation within membranes.
Acknowledgements by
We thank Dr. P.R. HiUiard for recording NMR spectra. This work was supported United States Public Health Service Research Grant HL 24312;
259
Grant HL 08214 from the Program Projects Branch, Extramural Programs, National Heart, Lung and Blood Institute; Center Grant NS 14304 from the National Institute of Neurological and Communicative Disorders and Stroke; and the Hormel Foundation. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
26 27 28
G.P. Hazlewood and R.M.C, Dawson, Biochem. J., 150 (1975) 521 -523. J.S. Ellingson, Biochemistry, 19 (1980) 6176-6182. R. Bomstein, Biochem. Biophys. Res. Commun., 21 (1965) 49-54. R.H. Quarles, N. Clarke and R.M.C. Dawson, Biochem. Biophys. Res. Comnlun., 33 (1968) 964-968. R.M.C. Dawson, N. Clarke and R.H. Quarles, Biochem. J., 114 (1969) 265 -270. G.M. Gray and H.J. Yardley, J. Lipid Res., 16 (1975) 441-447. G.M. Gray, Biochim. Biophys. Acta,431 (1976) 1 8. M. MatsumotoandM. Miwa, Biochim. Biophys. Acta, 296(1973) 350 364. V. Natarajan, P.C. Schmid, P.V. Reddy, M.L. Zuzarte-Augustin and H.tt.O. Schmid, Biochim. Biophys. Acta, 835 (1985) 426 433. P. Somerharju and O. Renkonen, Biochim. Biophys. Acta, 573 (1979) 83 89. D.E. Epps, V. Natarajan, P.C. Schmid and H.H.O. Schmid, Biochim. Biophys. Acta, 618 (1980) 420-430. V. Natarajan, P.C. Schmid and H.H.O. Schmid, Biochim. Biophys. Acta, 878 (1986) 32 -41. V. Natarajan, P.V. Reddy, P.C. Schmid and H.H.O. Schmid, Biochim. Biophys. Acta, 664 (1981) 445-448. V. Natarajan, P.V. Reddy, P.C. Schmid and H.H.O. Schmid, Biochim. Biophys. Acta, 712 (1982) 342-355. P.V. Reddy, V. Natarajan, P.C. Schmid and H.tt.O. Schmid, Biochim. Biophys. Acta, 750 (1983) 472-480. P.V. Reddy, P.C. Schmid, V. Natarajan and H.H.O. Schmid, Biochim. Biophys. Acta, 751 (1983) 241-246. V. Natarajan, P.C. Schmid, P.V. Reddy, M.L. Zuzarte-Augustin and H.H.O. Schmid, J. Neurochem., 42 (1984) 1613 1619. P.V. Reddy, P.C. Schmid, V. Natarajan, T. Muramatsu and H.H.O. Schmid, Biochim. Biophys. Acta, 795 (1984) 130-136. P.R. Cullis, A.J. Verkleij and P.H.J.Th. Ververgaert, Biochim. Biophys. Acta, 513 (1979) 11-20. S.B. Farren, M.J. Hope and P.R. Cullis, Biochem. Biophys. Res. Commun., 111 (1983) 675-682. D. Papahadjopoulos, W.J. Vail, K. Jacobson and G. Poste, Biochim. Biophys. Acta, 394 (1975) 483-491. P.H.J.Th. Ververgaert, B. de Kruijff, A.J. Verkleij, J.F. Tocanne and L.L.M. van Deenen, Chem. Phys. Lipids, 14 (1975)97-101. M.J. Hope and P.R. Cullis, FEBS Left., 107 (1979) 323-326. C.P.S. Tilcock and P.R. Cullis, Biochim. Biophys. Acta, 641 (1981) 189-201. P.R. Cullis, B. de Kruijff, M.J. Hope, A.J. Verkleij, R. Nayar, S.B. t:arren, C.P.S. Tilcock, T. Madden and M.B. Bally, in: R.C. Aloia (Ed.), Membrane Fluidity in Biology, Academic Press, 1983, pp. 39-81. A.J. Verkleij, Biochim. Biophys. Acta, 779 (1984) 43-63. P.R. Cullis and B. de Kruijff, Biochim. Biophys. Acta, 559 (1979) 399-420. D.A. Wilkinson and J.F. Nagle, Biochemistry, 20 ( 1981 ) 187 - 192.
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29 ('.P.S. T i l c o c k a n d P . R . C u l l i s , Biochim. Biophys. Acta, 6 8 4 ( 1 9 8 2 ) 212 218. 30 R.M. Epand, Chem. Phys. Lipids, 36 (1985) 3 8 7 - 3 9 3 . 31 P.W.M. van Dijck, P.H.J.Th. Ververgaert, A.J. Verkleij, L.UM. van Deenen and J. de Gier, Biochim. Biophys. Acta, 406 (1975) 4 6 5 - 4 7 8 . 32 D. Papahadjopoulos, W.J. Vail, C. Newton, S. Nit, K. Jacobson, G. Poste and R. Lazo, Biochim. Biophys. Acta, 465 (1977) 579 - 5 9 8 . 33 P.W.M. van Dijck, B. de Kruijff, L.LM. van Deenen, J. de Gier and R.A. Demel, Biochim. Biophys. Acta, 455 ~19761576 587. 34 P.R. Cullis, A.J. Verkleij and P.H.J.Th. Ververgaert, Biochim. Biophys. Acta, 513 (1978) 11 20.
249
I lsc~ier Scientific Publishers Ireland ltd.
PHASE BEHAVIOR OF SYNTHETIC N-ACYLETHANOLAMINE PHOSPHOLIPIDS
.I.1. NEWMAN*, D.I.. SI[IRS**, W.tl. ANI)I'2RSON and tt.tl.O. S('ItMID +
The tlormel Institute, Unirersity oJ Minnesota, Austin, MN 55912 (U.S.A.) Received .luly 2 l~t. 1986 revision received October 23rd, 1986 accepled October 23rd. 1986 Both saturated and unsaturated N-acylethanotamine phospholipids form lamellar structures t*hen dispersed in buffer. The addition of excess ('a 2÷ (('a2*/N-acytphosphatidylethanolamine > 0.5) results in precipitation. Vreeze-fracture replicas indicate that the addition of ('a 2÷ to the unsaturated lipid results in a non-bilayer structure ,~hile the ('a2+-complex of the saturated lipid is lamellar. Since unsaturated phospllatidylethanolaminc (PE) is a non-bilayer lipid, its N-acylalion with a saturated fatty acid converts a non-bilayer lipid into an acidic bilayer lipid capable of interacting with ('a ~÷ to return to a non-bilayer structure. ( a 2÷ may lhereby exert an influence on membrane phenomena by regulating phase behavior ~ithin certain membrane domains. Differential scanning calorimetry (DSC) indicates that N-acylation ol unsaturated Pt( ,s, ith a saturated fatty acid also results in changes in thermotropic phase behavior. Therefore, N-acylalion may affect fluidity ~ithin certain membrane domains.
Keywordsu N-acylphosphatidylethanolamine; phosphatidylelhanolamine; differential scanning calorimetry: freeze-fracture electron microscopy.
Introduction N - A c y l e t h a n o l a m i n e p h o s p h o l i p i d s o c c u r in m i c r o o r g a n i s m s [ 1 , 2 ] , p l a n t s 5] a n d certain v e r t e b r a t e tissues [6 9]. In the slime m o l d Dictyostelium discoideum, t h e y are present at a level o f a b o u t 10% o f t o t a l p h o s p h o l i p i d in the developing a m o e b a e b u t disappear d u r i n g t h e aggregation stage o f d e v e l o p m e n t [2]. They also disappear f r o m the seeds o f higher plants u p o n g e r m i n a t i o n [5]. In m a m m a l i a n cells, N - a c y l e t h a n o l a m i n e p h o s p h o l i p i d s can a c c u m u l a t e as the result of degenerative change, s u c h as t h a t o b s e r v e d in the granular cells of the e p i d e r m i s [3
*Present address: Worldwide tnnochem. Research and Development. S.(. Johnson & Son, Inc., 1525 llowe Street, Racine, Wl 53403, U.S.A. **Present address: Oklahoma Medical Research toundation, 825 NE 13th Street, Oklahoma City, OK 73104, U.S.A. +To whom correspondence should be addressed, Abbreviations: DS(', differential scanning calorimetry; HII, Hexagonal II; PC. phosphatidylcholine; PI-, phosphatidylethanolamine; constituent fatty acids are identified by parenthesis, e.g,, (dioleoyl)P[,;; the terms ethanolamine phospholipids and N-aeylethanolamine phospholipids indicate the actual or possible presence of diacyl, alkylacyl and alkenylacyl species, 000%3084/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
250 [6,7], in degenerating baby hamster kidney cells [10], in canine myocardial infarct [11] and in post-decapitative cerebral ischemia of the immature rat [12]. In both anaerobic bacteria [1] and mammalian tissues [13 18], N-acylethanolamine phospholipids are formed by acyl transfer to the amino group of ethanolamine phospholipids. In mammalian cells, this transfer involves 1-O-acyl g, oups of PE [14], phosphatidylcholine (PC) [15], and cardiolipin [16], and is catalyzed by a membrane-bound enzyme which exhibits an alkaline pH optimum and requires high amounts of Ca 2÷ for activity [ 13 -18]. Conversion of ethanolamine phospholipids to their N-acyl derivatives results not only in the addition of a hydrophobic moiety but also in the formation of an acidic phospholipid due to the loss of the free amino group. In order to evaluate the potential physiological or pathological significance of N-acylation reactions involving membrane phospholipids, it is of interest to determine the physical properties and phase behavior of N-acylated glycerophospholipids. The presence of Ca 2÷ has been shown to have a dramatic effect on the structures adopted by dispersions of acidic phospholipids and by mixed lipid systems containing them. Ca 2÷ complexes of some of these lipids, such as cardiolipin and phosphatidic acid, adopt hexagonal 11 (HII) phases [19,20], while others adopt various lamellar structures [21,22]. In liposomes composed of a mixture of acidic phospholipids with non-bilayer (HII) unsaturated PE, in which the bilayer structure is stable in the absence of Ca 2÷, the addition of Ca 2÷ triggers a bilayer to Hu transition [23,24]. The exact mechanism by which this occurs probably depends on the nature of the acidic phospholipid present [25]. Non-bilayer structures referred to as lipidic particles have been observed in these systems as probable intermediate structures in this process [26]. There is evidence for the existence of such structures within intact biological membranes as well. These lipidic particles, which have only been found in systems containing HII lipids, have been postulated to play a role in membrane fusion events, such as endocytosis and exocytosis, as well as in transbilayer transport phenomena [25 27]. Thus, the ability of Ca 2+ to modulate the occurrence of these lipidic particles may provide a means of regulation of certain membrane processes. Conversion of PE, a non-bilayer lipid, to N-acyl-PE may provide a mechanism for isothermal control over the occurrence of lipidic particles and thus over fusion of membrane transport processes. In this paper, we report the phase behavior of saturated and unsaturated N-acyl-PE using DSC and freeze-fracture electron microscopy.
Experimental N-Acyl-PE synthesis 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine was obtained from Calbiochem:
1,2-dipalmitoyl- and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine were obtained
251 from Avanti. Fatty acids were purchased from Sigma. 1,2-Dilauroyl-sn-glycero-3phospho(N-lauroyl)ethanolamine and 1,2-dipahnitoyl-or 1,2-dioleoyl-sn-glycero-3phospho(N-palmitoyl~ethanolamine were synthesized by acylation of (dilauroyl)PE, (dipalmitoyl)PE, and (dioleoyl)PE, respectively. The fatty acid (2 mmol) was reacted with N,N'-carbonyldiimidazole (1.8 mmol) in dry benzene for 4 h to form the fatty acid imidazolide. The PE (1 mmol) was then added to the reaction mixture which was stirred overnight. Thin-layer chromatography on silica gel H plates (chloroform/methanol/conc. ammonium hydroxide, 80:20:2, by vol.)showed that the reaction was essentially complete. The synthetic N-acyl-PE was purified by silicic acid column chromatography. The crude product was applied to the column in chloroform and chloroform or chloroform/methanol mixtures were used for elution. The pure N-acyl-PE was eluted with chloroform/methanol (90: 10, v/v). Thin-layer chromatography of a heavily-loaded sample revealed no detectable impurities. The migration rate of the purified product was identical to that of N-acyl-PE previously synthesized and characterized [9,11 18].
Preparation of lipid dispersions A chloroform solution of the lipid was evaporated to dryness under a stream of nitrogen. Residual solvent was removed by drying the lipid under vacuum overnight. The saturated phospholipids were then dispersed in 100 mM NaC1, 50 mM Tris HC1 (pH 7.2) by extensive vortexing at or above 55°C, whereas, the unsaturated lipid was vortexed at room temperature. The buffer was diluted to 20% glycerol in samples for freeze-fracture to prevent freeze damage or to 40% ehtylene glycol for calorimeter samples when scanning from sub-zero temperatures was desired. To obtain samples with the desired Ca2+/N-acyl-PE molar ratios, appropriate aliquots of a 100 mM or 200 mM CaCI 2 stock solution were added.
DSC DSC thermograms were obtained using a MicroCal MC-2 differential scanning calorimeter. The sample cell contained 1.22 ml of the lipid dispersion (2.5-7.5 ~mol/ml or 2 6 mg/ml), while the reference cell held the same volume of the buffer used in the dispersion. Heating scans were performed at a scanning rate of 10° or 20°C/h.
Freeze-fracture electron microscopy Dispersions or precipitates were transferred to specimen holders equilibrated at the desired temperatures and were then rapidly quenched in liquid freon or liquid propane at liquid nitrogen temperature. Replicas were prepared by standard freeze-fracture techniques with a Balzers BAF 300 apparatus. Replicas were floated off in water and cleaned with chloroform, when necessary. Micrographs of replicas were obtained with a J EOL 100-S electron microscope.
252
Results and Discussion When vortexed at the appropriate temperatures, both the saturated and unsaturated N-acyl-PE formed milky dispersions. In contrast, (dioleoyl)PE, which is a non-bilayer lipid, would not form a stable homogeneous dispersion at room temperature. When excess Ca > (Ca>/N-acyI-PE > 0.5) was added to the concentrated dispersions of either the saturated or unsaturated N-acyl-PE used in freeze-fracture experiments, a precipitate formed, as is common with other acidic phospholipids. In the less concentrated calorimeter samples, the formation of a precipitate was not immediately apparent. Therefore, it appears that the effect of Ca =+ is dependent on the actual lipid or cation concentration as well as 1he Ca2+/N acyl-PE ratio.
Calorimetry DSC thermograms of N-lauroyl(dilauroyl)PE reveal a narrow energetic endotherm at 32°C indicative of a gel-to-liquid-crystal transition (Fig. 1 ). The transition half-width measured 0.3°C in this heating scan obtained at a heating rate of IO°('ih. This value is comparable to those obtained for saturated PE [28]. The transition temperature of the parent compound is 30.5°C, very close to that of the N-acylated derivative [28]. Tile gel-to-liquid-crystal transition of N-pahnitoyl(dipahnitoyl)Pl,~ also occurs at approximately the same temperature as that of the parent compound, (dipalmitoyl)PE (data not shown). The fact that N-acylation of saturated PE with the same fatty acid does not effect a change in the transition temperature indicates that the change in head group region of the lipid has a negligible effect on the transition temperature of the lipid. Figure 2 shows a series of thermograms obtained after addition of excess ('a 2+ to N-lauroyl(dilauroyl)PE. In the first scan (A), the original peak at about 32°( ' is present as well as an additional endotherm at 380( ". Successive heating scans (B and C) reveal an increase in the enthalpy of the 380( ` transition concomitant with a decrease in the enthalpy associated with the 32°( ` transition. In the third scan of the series (CL the lower-temperature transition is no longer present. Addi, tionally, two unresolved peaks can be seen at about 3~)° and 41°C. These resulis suggest that the Ca>-complex melts at a higher temperature than does the free lipid and is apparently formed at higher temperatures. This may be indicative of limited initial accessibility of the acidic lipid head groups to external Ca 2+. The heating scan for commercial (dioleoyl)PE (Avanti) indicates that the reversible gel-to-liquid-crystal transition occurs at 6°C (data not shown). The transition half-width of 0.8°C for this unsaturated lipid is greater than that obtained for saturated PE and N-acyl-PE. This may be due in part to the higher heating rate of 20°C/h required for scanning from sub-zero temperatures on the MC-2 instrument. While previous NMR studies have shown that a bilayer ---* ttll transition occurs between 7°C and 12°C in this PE [291, no endotherm other than
253
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the main transition is apparent in this scan. However, a relatively weak exotherm at 0°C can be seen when the sensitivity is increased. The detection of the bilayer HII transition using higher sensitivity DSC has recently been reported [30]. The enthalpy of this transition was dependent upon the length of time the lipid was incubated at 0°C. This phenomenon may be related to the exotherm observed in our study at 0°C. The synthetic N-palmitoyl derivative of (dioleoyl)PE exhibits its main endotherm at +2°C with a slight shoulder at about 0°C (Fig. 3). Therefore, N-acylation of (dioleoyl)PE with saturated palmitic acid results in an increase in the gel-toliquid-crystal transition by 8°C. Thus, it would appear that fluidity within membrane domains could potentially be affected by N-acylation of PE, depending on the nature of the acyl chain involved. As is the case with the saturated N-acyl-
254
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Fig. 2. ConsecutiveDSC scans(A,B and C)ofadispersion ofN-lauroyl(dilauroyl)PEin buffer with e×cessCa2+(Caa+/N~cyl2E = 0.6)ata heatingrate oflO°C/h.
PE, the addition of excess Ca 2+ to this unsaturated N-acyl-PE results in an increase in the transition temperature to 6°C, as shown in the successive thermograms in Fig. 4. A minor exotherm at 0°C can also be seen in these scans.
Freeze-fracture Electron micrographs of freeze-fracture replicas of N-lauroyl(dilauroyl)PE dispersions at temperatures up to 65°C reveal liposomal structures (Fig. 5), while
255
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Fig. 3. DSC scan of a dispersion of N-palmitoyl(dioleoyl)PE in buffer plus 40% ethylene glycol at a heating rate of 20"C/h.
replicas of the Ca 2+ complex of this lipid indicate a stacked lamellar structure (Fig. 6). These results are similar to those obtained for saturated phosphatidylglycerol [31] and phosphatidytserine [32]. Quenching from temperatures above 30°C may not prevent a possible transition from an HII to a lamellar phase [26]. Thus, these freeze-fracture results are not sufficient to determine the correct phase of this lipid at 45°C. However, when dispersions of the lipid (in the absence of Ca 2+) were heated to temperatures in excess of 65°C, no precipitate indicative of the HII phase was observed, and 31P-NMR spectra indicated lamellar structure for this lipid and the Ca 2÷ complex up to 45°C. Replicas of N-palmitoyl(dioleoyl)PE dispersions at room temperature up to 55~C show a lamellar liposomal structure (Fig. 7) as opposed to the HII of (dioleoyl)PE [33]. Although freeze-fracture data cannot definitively identify lamellar structures above 30°C, these results indicate that this lipid remains in the bilayer form up to 30°C. Because the N-acylated derivative of a non-bilayer
256
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TEMPERATURE (oc) Fig. 4. Consecutive DSC scans o f a dispersion of N-palmitoyl(dioleoyl)PE in buffer plus 40% ethylene glycol with excess Ca 2+ (Ca2+/N-acyl-PE = 0.6) at a heating rate o f 20*C/h.
Fig. 5. Electron micrograph of a freeze-fracture replica of an N-lauroyl(dilauroyl)PE dispersion in buffer plus 20% glycerol at 45"C.
257
Fig. 6. Electron micrograph of a freeze-fracture replica of the Ca2+-complex of N-palmitoyl(dioleoyl)PE at 45°C, formed by the addition of excess Ca 2÷ (Ca2"/N-acyl-PE = 0.6) to a dispersion of the lipid in buffer plus 20% glycerol.
Fig. 7. Electron micrograph of a freeze-fracture replica of a dispersion of N-palrnitoyl(dioleoyl)PE in buffer plus 20% glycerol at 45"C.
258
Fig. 8. Electron micrograph of a freeze fracture replica of the Ca2÷-complex of N-palmitoyldioleoyl)PE at 55"C, formed by the addition of excess Ca2÷ (Ca2÷/N-acyl-PE= 0.6) to a dispersion of the lipid in buffer plus 20% glycerol.
lipid forms lamellar structures, N-acylation may have an overall bilayer-stabilizing effect in certain membrane domains. The structure of the Ca 2÷ complex of this unsaturated N-acyl-PE was also investigated by the freeze-fracture technique. As is the case with cardiolipin [34], Ca 2÷ appears to convert the saturated form of this acidic phospholipid to a HII phase (Fig. 8). Thus, although N-acylation converts the non-bilayer unsaturated PE into a bilayer lipid, the addition of Ca 2÷ can reverse the bilayer-stabilizing effect. These results suggest that N-acylation o f unsaturated PE can have a bilayerstabilizing effect within membrane domains. In addition, Ca 2÷ is capable of modulating this stabilizing effect. This could possibly affect the occurrence of nonbilayer structures, such as lipidic particles and could thereby affect membrane fusion and transport phenomena. Depending on the nature of the added acyl group, N-acylation could also have an effect on membrane fluidity in certain domains. Further studies of mixed lipid systems containing N-acyl-PE, PE and other lipids, should reveal more about the potential effects of N-acylation within membranes.
Acknowledgements by
We thank Dr. P.R. HiUiard for recording NMR spectra. This work was supported United States Public Health Service Research Grant HL 24312;
259
Grant HL 08214 from the Program Projects Branch, Extramural Programs, National Heart, Lung and Blood Institute; Center Grant NS 14304 from the National Institute of Neurological and Communicative Disorders and Stroke; and the Hormel Foundation. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
26 27 28
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