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Unusual pressure dependence of the lateral motion of pyrene-labeled phosphattdylcholine in bipolar lipid vesicles
Vol.
188,
No.‘~,
NoveAber
16,
1992
BIOCHEMICAL
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COMMUNICATIONS
Pages
1992
1241-1246
UNUSUAL PRESSURE DEPENDENCE OF THE LATERAL MOTION OF PYRENE-LABELED
PHOSPHATIDYLCHOLINE
IN BIPOLAR LIPID VESICLES
Yvonne L. Kao*, Eddie L. Char+, and Parkson L.-G. Chongl.#.
1Dept. of Biochemistry, Meharry Medical College, Nashville, TN 37208 ‘Xenter for BioMolecular Science and Engineering, Code 6090, Naval Research Laboratory, Washington, D.C. 203755320 Received
October
1, 1992
Summary: The lateral mobility of a pyrene-labeled phosphatidylcholine probe in liposomes containing archaebacterial bipolar lipids has been studied isothermally as a function of pressure. The pressure-dependence of the probe mobility, R, is found to be slightly positive or zero in the temperature range of 17 - 48 “C. At temperatures > 48 “C, R becomes negative and decreases with temperature. The data indicate that lateral mobility only becomes appreciable at high temperatures. In addition, the R values obtained with other lipid membranes are much lower than that obtained with bipolar liposomes, implying that the membranes of archaebacterial liposomes are laterally immobile, as compared to other lipid membranes. %‘1992Academic Press,Inc.
Sulfolobw acidoculdurius is a thermoacidophilic archaebacterium found in hot (65 - 80 “C) and acidic sulfur springs. The membrane of S. acidocaldurius consists mainly of bipolar tetraether lipids (nearly 90% of the total lipid) that have the potential to span the membrane (1). PLFE, the major polar lipid fraction in S. ucidoculdurius (PLFE is the S. ucidoculdurius equivalent to the P2 fraction from another thermophile S. soljururicus ), has phosphatidylinositol as one polar group and either glucopyranose or galactopyranosyl glucopyranose disaccharide as the other polar group (1,2) (Figure 1). The physiological significance of these unique lipid structures in S. ucidoculdurius is, however, not well understood. Physical studies to answer these questions can now be done with the recent demonstration that PLFE lipids can spontaneously form multilamellar liposomes in aqueous media (3).
#To whom correspondence should be addressed at Department of Biochemistry, Meharry Medical College, Nashville, TN 37013. Fax: (615) 327-6442. Abbreviations. C(18):C(lO)PC, l-stearoyl-2-capryl-sn-glycero-3-phosphocholine; diphyPC, diphytanoylphosphatidylcholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DSC, differential scanning calorimetry; egg-PC, egg yolk phosphatidylcholine; FRAP, fluorescence recovery after photobleaching; PLFE, polar lipid fraction E; PGPC, 1-palmitoyl-2-oleoylphosphatidylcholine; PyrPC, 1-palmitoyl-Z( lo-pyrenyl)docanoyl)-sn-glycero-3phosphatidylcholine
1241
0006-291 X/92 $4.00 Copyright 0 1992 by Academic Press, Iw. All rights of reproduction in any form reserved
Vol.
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a OHOHOH
Figure 1.
b OHOH
The structures of the lipid mixture PLFE consist of (a) a digycerol skeleton with
phosphatidylmyoinositol(PI) attachedto one glycerol and Rl = P-D-gal-p-Dglc-p and (b) a glyceroI/nonitol skeletonwith PI on one end and R2=gD-glc-p. The actual number of cyclopentanerings variesfrom 0 to 8.
Elucidation of the functional role of archaebacterial lipids requires an understanding of their static and dynamic properties in membranes (4). Vaz et al. (5) have previously used fluorescence recovery after photobleaching (FRAP) techniques to measure the translational diffusion of a fluorescently-labeled, hydrolyzed (i.e., no polar head groups) bipolar lipid prepared from S. solfatticus in POPC multilamellar vesicles in the liquid-crystalline state. They showed that the membrane-spanning archaebacterial tetraether lipid probe had a translational diffusion coefficient -2/3 that of lipids which extend to only one monolayer of the bilayer. The lateral diffusion coefficient, as determined by FRAP, provides a measure of macroscopic and long-range property, which, however, may be significantly different from the microscopic lateral diffusion coefficient (6,7) determined from other techniques such as excimer formation method (8). Since lateral mobility is one of the most important properties in a functional biomembrane, it is of interest to characterize the microscopic lateral mobility of a lipid probe in the unusual environment of a bipolar archaebacterial lipid membrane. In the present study, we examine the effects of pressure (0.001 - 1.5 kbar) and temperature (15 - 60 “C), at pH 7.4, on the microscopic lateral mobility of PyrPC in liposomes composed of PLFE. While S. aciubcaldarius grow optimally at pH 2, the internal pH of acidophiles, includiig sulfolobur, is generally around pH 6 (9,lO). Further, there is evidence that the bipolar lipids are distributed asymmetrically in the native membrane with most of the negatively-charged phosphate headgroup facing inside (11). Therefore, measurements made under conditions where the negative lipid headgroup is fully charged should closely reflect an important aspect of the actual lipid state. Pressure is used here as a tool to modulate the lateral mobility of PyrPC in liposomes. Previous pressure studies using pyrene and its derivatives in membranes have shown that increased pressure causes a decrease in the lateral motion of the probe (12-15). Our present data indicate an unusually low lateral-mobility of lipids in PLFE liposomes. These results provide a starting point for future work on lipid-protein interactions in bipolar lipid membranes. 1242
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Materials - PLFE was isolated from S. acidocaldarius grown at 65-67 “C using the revised method of reverse-phase column and methanol precipitation (3). C( 18):C( 10)PC was kindly provided by Professor Ching-hsien Huang at the University of Virginia. DMPC, PGPC, and diphyPC were obtained from Avanti Lipids (Birmingham, AL). PyrPC was obtained from KSV Chemicals (Kauniainen, Finland). Liposome Preparation- PyrPC dissolved in hexane was first dried under nitrogen in a test tube. PLFE was then added and co-lyophilized with PyrPC in a mixture of chlorofornnmethanol: water (65/25/10) (3). When other lipids were used as the matrix lipids, they were co-lyophilized with PyrPC in hexane. The lipids were suspended in 20 mM PIPES, 10 mM NaCl, 0.02% NaNs (pH 7.4) and vortexed above the melting transition temperature, Tm @SC did not reveal any Tm for PLFE liposomes over the temperature range of 5 ‘C - 90°C--Chang, unpublished results), of the matrix lipid to form multilamellar vesicles. The PyrPC/C( 18):C( 10)PC mixture was put through three freeze-thaw cycles and then stored at 4 ‘C for at least 48 hours to remove inhomogeneous mixing. PLFE vesicles were prepared at 65 ‘C. Lipid concentrations, determined by the method of Bartlett (16), used for fluorescence measurements were about 1 mM. Pyrene concentrations were determined using the extinction coefficient of 50,000 cm-tM-1. Fluorescence Measurements under Pressure - Fluorescence emission spectra were measured with an ISS Greg 200 fluorometer (ISS Inc., Champaign, IL) and an SLM DMX-1000 fluorometer (SLM-Aminco, Urbana, IL). The samples were excited at 325 nm using a xenon arc lamp. The procedures for measurement of spectra under pressure have been previously described (17). An ISS-Nova pressure cell (ISS Inc.) and an SLM high pressure cell (Urbana, IL) were used for the experiments. The excimer and monomer wavelengths were chosen at 480 nm and 395 nm respectively. All the pressure measurements were done under isothermal conditions. To examine whether any free PyrPC was unincorporated into PLFE, we quantitated the amounts of fluorescence by separating the liposomes on a Sepharose CL-2B column at ambient temperature. It was found that the PyrPC (as detected by fluorescence) eluted in the same fraction as that of the matrix lipid (as determined by the inorganic phosphate assay (16)). No second peak of PyrPC micelles or liposomes was found. This indicates that almost all the PyrPC molecules are associated with the matrix lipid.
Results
and Discussion
R, defined as the change in excimer/monomer formation per kilobar, A(E/M)ikbar, is used as an index of the pressure sensitivity of the lateral mobility of PyrPC in PLFE membranes. A(E/M) is determined from the initial slope in the plot of E/M vs. pressure. If PyrPC were laterally mobile, then pressure should reduce the mobility, and thereby E/M, yielding an appreciably negative R value. If PyrPC were completely immobile in the medium, then pressure would not cause a decrease in lateral mobility, and, thus no changes in E/M by pressure (R=O). R, in the case of PLFE liposomes, is unlikely to be affected by the pressure dependence of the excimer lifetime, rn. Turley and Gffen (14) have shown that 7~ of dipyrene propane is virtually invariant with pressure when the matrix lipid is in the disordered state and that rn increases with pressure only when the matrix lipid becomes ordered. Since the packing of the PLE liposomes is likely to be disordered (Chang, unpublished FT-IR results), rn is not expected to vary with pressure. Figures 2 shows the effect of pressure on the fluorescence intensity ratio (F48O/F395) for PyrPC in the PLFE liposomes (1:500). The slope of F48O/F395 vs. pressure is positive between 16.9’C to 47.5 “C. At 47.5 ‘C, the slope is zero, and by 56.6 ‘C F48O/F395 starts to decrease with pressure. R drops from a slightly positive 0.0053 at 16.9”C to a value of -0.0053 at 56.6 “C. 1243
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Figure 2.
1.5
2.0
(kbar)
F480/F395 for PyrPCin PLFE liposomesasa function of temperatureand pressure. A.(O)=16.9 ‘C;(O)= 26.9 “C; and (0) = 37.6 “C. B. A = 47.5 ‘C and (A) = 56.6.V. [PLFE]/[PyrPC] = 500/l.
Positive values of R are not observed for any other lipids. The zero R-value at 48 ‘C suggests that at this temperature PyrPC is virtually laterally immobile in the PLPE liposomes and starts gaining some lateral mobility only for T > 48 “C. The positive R-values observed for T c 48 ‘C are rather surprising. This anomaly may be interpreted within the context of “monomer pairs” (1X,19), which predicts that excimers of pyrene will form only if the pyrene rings of the neighboring monomer pairs are oriented in an appropriate direction, presumably in a parallel disposition. In the temperature range of 15 - 48 ‘C, PLFE liposomes appear to be very rigid. Pressure probably has little infhtence on the lateral mobility of PyrPC. However, in such a rigid environment, pressure may alter the mutual orientation of the pyrene rings between the neighboring pyrene monomer pairs, forcing the neighboring pyrene rings to align parallel to each other. As a result, the E/M increases with pressure, giving the positive anomalous R values. According to this explanation, both R = 0 and R > 0 can be considered as indicating that no significant lateral mobility exists for the probe. The R values of all the lipid systems examined are summarized in Figure 3. Branched methyl-groups in diphyPC significantly affect interchain packing and lipid dynamics due to steric effects (20-22). Stewart et al. (20) have previously shown that increased branching decreases the 1244
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Temperature dependence of R in various lip&omes. R is the change in F48O/F395 perkbar. PLFE (1:SOO); PLFE (1:X)); POPC (1:SOO); diphyPC (1:500); C(18):C(lO)PC (1:500); and DMPC (1:250). All F48O/F395 values were normalized to a PyrPCxnatrix lipid molar ratio of 1:500.(O)= PLFE (1:500),(e)= PLFE (1:50),(x)= DMPC, (m) = DiphyPC, (Cl) = POPC, and(A)= C(18):C(lO)PC.
membrane order. This strongly suggests that branching can be a way to maintain the membranes in a disordered state over a wide temperature, pressure, and pH range. Our results shown in Figure 3 indicate that the R values for diphyPC, while higher than for straight-chain lipids, are more negative than for PLFR, suggesting that the low lateral-mobility of PyrPC in PLFE liposomes cannot be wholly attributed to branched methyl groups. C( lS):C( lO)PC, which forms mixed interdigitated structures below T,,, (18.8 “C) and partially interdigitated structures above T,,, was used to test whether the tightly packed, interdigitated state would present a similar matrix environment as PLFE. Also, in the mixed interdigitated form, one of the two acyl chains interpenetrates across the entire membrane, resembling the cross-linked structure of PLFEZ. However, it is seen in Figure 3 that PyrPC in C( 1S):C( 10)PC exhibits considerable lateral mobility. Thus, it is also unlikely that the low lateral mobility of PyrPC in PLFE liposomes can be attributed to monomolecular packing alone. While lateral mobility of PyrPC in PLFE liposomes is affected by the probe:lipid ratio (Figure 3), previous studies suggested that PyrPC does not form segregated domains in liposomes in the probe concentrations examined (0.2 mol% - 2 mol%) (6,24,25). Also, epifluorescence microscopy observations of the fluorophore distribution of N-lissamine-rhodamine B sulphonyl diacyl phosphatidylethanolamine (1 mol%) in PLFE do not show probe phase-separation (private communication with Dr. A. Gliozzi). If this situation holds true for PyrPC in PLFE liposomes, then the decrease in R as probe concentration increases can be ascribed to probe-induced increase in membrane perturbation, which increases the lateral mobility of PyrPC. When the temperature for which R = 0 is extrapolated to the case of zero probe-concentration, then R = 0 at about 58 ‘C for pure PLFE lipids, which is close to the minimum gxowth temperature of S. acidoculdurius . This implies that PLPE liposomes begin to gain fluidity at T > 58 OCand may be taken to explain partially why S. acidocaldarius is adapted to living at high temperatures, 1245
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Acknowledgments This work was supported by the NSF-RIM1 (RII-9014052). This work was done during the tenure of an Established Investigatorship (to P. L.-G. C.) from the American Heart Association and CIBA-GEIGY. E.L.C. would like to acknowledge support from the Office of Naval Research, through the Core Program at Naval Research Laboratory.
References 1. 2 i: 5. 6. 7. 8. 9. 10. 11 12. 13. 14. 15. 16. 17. 18. :;: 2 23: 24. 25.
Langworthy, T. A. (1985) In Archaebacteria VIII (Woese, C. R. andR.S. Wolfe, R. S., Eds.). pp.459-498 Academic Press, NYC, NY De Rosa, M., Gambacorta, A., Gliozzi, A. (1986) Microbiological Reviews 50,70-80 Lo, S.-L., and Chang, E. L. (1990) B&hem. Biophys. Res. Comm. 167,238 - 243 Luzzati, V., Gulik, A., DeRosa, M., and Gambacorta, A. (1987) Chemica Scripta 27B, 211-219 Vaz, W. L. C., Hallmann, D., Clegg, R. M., Gambacorta, A., and DeRosa, M. (1985) Eur. Biophys. J. 12, 19 - 24 Chong, P. L.-G., and Thompson, T.E. (1985) Biophys. J. 47,613-621 Vaz, W. L. C., and Almeida, P. F. (1991) Biophys. J. 60, 1553-1554 Galla, H.-J., Hartmann, W., Theilen, U. and Sackmann, E. (1979) J. Membr. Biol. 48, 215-236 Cobley, J.G. and Cox, J.C. (1983) Microbiological Reviews 47, 579-595 Krulwich, T.A. and Guffanti, A.A. (1983) Advances in Microbial Physiology 24, 173214 DeRosa, M., Gambacorta, A. and Nicolaus, B. (1983) Journal of Membrane Science 16, 287-297 Flamm, M., Okubo, T., Turro, N. J., and Schachter, D. (1982) B&him. Biophys. Acta 687,101 - 104 Mtiller, H. R., and Calla, H. H. (1983) Biochim. Biophys. Acta 783,291 - 294 Turley, W. D., and Offen, H. W. (1986) J. Phys. Chem. 90, 1967 - 1970 Macdonald, A. G., Wahle, K. W. J., Cossins, A. R., and Behan, M. K. (1988) Biochim Biophys. Acta 938,231 - 242 Bartlett, G. R. (1959) J. Biol. Chem. 234,466 - 468 Chong, P. L.-G., and Weber, G. (1983) Biochemistry 22,5544 - 5550 Sugar, I. P., Zeng, J., Vauhkonen, M., Somerharju, P., and Chong, P. L.-G. (1991a) J. Phys. Chem. 95,7516 - 7523 Sugar, I. P., Zeng, J., and Chong, P. L.-G. (1991b) J. Phys. Chem. 95,7524 - 7534 Lindsey, H., Petersen, N.O., and Chan, S.I. (1979) B&him. Biphys. Acta 555, 147167 Plachy, W. Z., Lanyi, L. K., and Kates, M. (1974) Biochemistry 13,4906 - 4913 Jackson, M. B., and Sturtevant, J. M. (1978) Biochemistry 17,4470-4474 Stewart, L. C., Kates, M., Ekiel, I. H., and Smith, I. C. P. (1990) Chem. Phys. Lipids 54, 115 - 129 Roseman, M. A., and Thompson, T. E. (1980) Biochemistry 19,439-444 Hresko, R. C., Sugar, I. P., Barenholz, Y. and Thompson, T. E. (1986) Biochemistry 25,3813-3823
1246
188,
No.‘~,
NoveAber
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Pages
1992
1241-1246
UNUSUAL PRESSURE DEPENDENCE OF THE LATERAL MOTION OF PYRENE-LABELED
PHOSPHATIDYLCHOLINE
IN BIPOLAR LIPID VESICLES
Yvonne L. Kao*, Eddie L. Char+, and Parkson L.-G. Chongl.#.
1Dept. of Biochemistry, Meharry Medical College, Nashville, TN 37208 ‘Xenter for BioMolecular Science and Engineering, Code 6090, Naval Research Laboratory, Washington, D.C. 203755320 Received
October
1, 1992
Summary: The lateral mobility of a pyrene-labeled phosphatidylcholine probe in liposomes containing archaebacterial bipolar lipids has been studied isothermally as a function of pressure. The pressure-dependence of the probe mobility, R, is found to be slightly positive or zero in the temperature range of 17 - 48 “C. At temperatures > 48 “C, R becomes negative and decreases with temperature. The data indicate that lateral mobility only becomes appreciable at high temperatures. In addition, the R values obtained with other lipid membranes are much lower than that obtained with bipolar liposomes, implying that the membranes of archaebacterial liposomes are laterally immobile, as compared to other lipid membranes. %‘1992Academic Press,Inc.
Sulfolobw acidoculdurius is a thermoacidophilic archaebacterium found in hot (65 - 80 “C) and acidic sulfur springs. The membrane of S. acidocaldurius consists mainly of bipolar tetraether lipids (nearly 90% of the total lipid) that have the potential to span the membrane (1). PLFE, the major polar lipid fraction in S. ucidoculdurius (PLFE is the S. ucidoculdurius equivalent to the P2 fraction from another thermophile S. soljururicus ), has phosphatidylinositol as one polar group and either glucopyranose or galactopyranosyl glucopyranose disaccharide as the other polar group (1,2) (Figure 1). The physiological significance of these unique lipid structures in S. ucidoculdurius is, however, not well understood. Physical studies to answer these questions can now be done with the recent demonstration that PLFE lipids can spontaneously form multilamellar liposomes in aqueous media (3).
#To whom correspondence should be addressed at Department of Biochemistry, Meharry Medical College, Nashville, TN 37013. Fax: (615) 327-6442. Abbreviations. C(18):C(lO)PC, l-stearoyl-2-capryl-sn-glycero-3-phosphocholine; diphyPC, diphytanoylphosphatidylcholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DSC, differential scanning calorimetry; egg-PC, egg yolk phosphatidylcholine; FRAP, fluorescence recovery after photobleaching; PLFE, polar lipid fraction E; PGPC, 1-palmitoyl-2-oleoylphosphatidylcholine; PyrPC, 1-palmitoyl-Z( lo-pyrenyl)docanoyl)-sn-glycero-3phosphatidylcholine
1241
0006-291 X/92 $4.00 Copyright 0 1992 by Academic Press, Iw. All rights of reproduction in any form reserved
Vol.
188, No. 3, 1992
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AND BIOPHYSICAL
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a OHOHOH
Figure 1.
b OHOH
The structures of the lipid mixture PLFE consist of (a) a digycerol skeleton with
phosphatidylmyoinositol(PI) attachedto one glycerol and Rl = P-D-gal-p-Dglc-p and (b) a glyceroI/nonitol skeletonwith PI on one end and R2=gD-glc-p. The actual number of cyclopentanerings variesfrom 0 to 8.
Elucidation of the functional role of archaebacterial lipids requires an understanding of their static and dynamic properties in membranes (4). Vaz et al. (5) have previously used fluorescence recovery after photobleaching (FRAP) techniques to measure the translational diffusion of a fluorescently-labeled, hydrolyzed (i.e., no polar head groups) bipolar lipid prepared from S. solfatticus in POPC multilamellar vesicles in the liquid-crystalline state. They showed that the membrane-spanning archaebacterial tetraether lipid probe had a translational diffusion coefficient -2/3 that of lipids which extend to only one monolayer of the bilayer. The lateral diffusion coefficient, as determined by FRAP, provides a measure of macroscopic and long-range property, which, however, may be significantly different from the microscopic lateral diffusion coefficient (6,7) determined from other techniques such as excimer formation method (8). Since lateral mobility is one of the most important properties in a functional biomembrane, it is of interest to characterize the microscopic lateral mobility of a lipid probe in the unusual environment of a bipolar archaebacterial lipid membrane. In the present study, we examine the effects of pressure (0.001 - 1.5 kbar) and temperature (15 - 60 “C), at pH 7.4, on the microscopic lateral mobility of PyrPC in liposomes composed of PLFE. While S. aciubcaldarius grow optimally at pH 2, the internal pH of acidophiles, includiig sulfolobur, is generally around pH 6 (9,lO). Further, there is evidence that the bipolar lipids are distributed asymmetrically in the native membrane with most of the negatively-charged phosphate headgroup facing inside (11). Therefore, measurements made under conditions where the negative lipid headgroup is fully charged should closely reflect an important aspect of the actual lipid state. Pressure is used here as a tool to modulate the lateral mobility of PyrPC in liposomes. Previous pressure studies using pyrene and its derivatives in membranes have shown that increased pressure causes a decrease in the lateral motion of the probe (12-15). Our present data indicate an unusually low lateral-mobility of lipids in PLFE liposomes. These results provide a starting point for future work on lipid-protein interactions in bipolar lipid membranes. 1242
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Materials - PLFE was isolated from S. acidocaldarius grown at 65-67 “C using the revised method of reverse-phase column and methanol precipitation (3). C( 18):C( 10)PC was kindly provided by Professor Ching-hsien Huang at the University of Virginia. DMPC, PGPC, and diphyPC were obtained from Avanti Lipids (Birmingham, AL). PyrPC was obtained from KSV Chemicals (Kauniainen, Finland). Liposome Preparation- PyrPC dissolved in hexane was first dried under nitrogen in a test tube. PLFE was then added and co-lyophilized with PyrPC in a mixture of chlorofornnmethanol: water (65/25/10) (3). When other lipids were used as the matrix lipids, they were co-lyophilized with PyrPC in hexane. The lipids were suspended in 20 mM PIPES, 10 mM NaCl, 0.02% NaNs (pH 7.4) and vortexed above the melting transition temperature, Tm @SC did not reveal any Tm for PLFE liposomes over the temperature range of 5 ‘C - 90°C--Chang, unpublished results), of the matrix lipid to form multilamellar vesicles. The PyrPC/C( 18):C( 10)PC mixture was put through three freeze-thaw cycles and then stored at 4 ‘C for at least 48 hours to remove inhomogeneous mixing. PLFE vesicles were prepared at 65 ‘C. Lipid concentrations, determined by the method of Bartlett (16), used for fluorescence measurements were about 1 mM. Pyrene concentrations were determined using the extinction coefficient of 50,000 cm-tM-1. Fluorescence Measurements under Pressure - Fluorescence emission spectra were measured with an ISS Greg 200 fluorometer (ISS Inc., Champaign, IL) and an SLM DMX-1000 fluorometer (SLM-Aminco, Urbana, IL). The samples were excited at 325 nm using a xenon arc lamp. The procedures for measurement of spectra under pressure have been previously described (17). An ISS-Nova pressure cell (ISS Inc.) and an SLM high pressure cell (Urbana, IL) were used for the experiments. The excimer and monomer wavelengths were chosen at 480 nm and 395 nm respectively. All the pressure measurements were done under isothermal conditions. To examine whether any free PyrPC was unincorporated into PLFE, we quantitated the amounts of fluorescence by separating the liposomes on a Sepharose CL-2B column at ambient temperature. It was found that the PyrPC (as detected by fluorescence) eluted in the same fraction as that of the matrix lipid (as determined by the inorganic phosphate assay (16)). No second peak of PyrPC micelles or liposomes was found. This indicates that almost all the PyrPC molecules are associated with the matrix lipid.
Results
and Discussion
R, defined as the change in excimer/monomer formation per kilobar, A(E/M)ikbar, is used as an index of the pressure sensitivity of the lateral mobility of PyrPC in PLFE membranes. A(E/M) is determined from the initial slope in the plot of E/M vs. pressure. If PyrPC were laterally mobile, then pressure should reduce the mobility, and thereby E/M, yielding an appreciably negative R value. If PyrPC were completely immobile in the medium, then pressure would not cause a decrease in lateral mobility, and, thus no changes in E/M by pressure (R=O). R, in the case of PLFE liposomes, is unlikely to be affected by the pressure dependence of the excimer lifetime, rn. Turley and Gffen (14) have shown that 7~ of dipyrene propane is virtually invariant with pressure when the matrix lipid is in the disordered state and that rn increases with pressure only when the matrix lipid becomes ordered. Since the packing of the PLE liposomes is likely to be disordered (Chang, unpublished FT-IR results), rn is not expected to vary with pressure. Figures 2 shows the effect of pressure on the fluorescence intensity ratio (F48O/F395) for PyrPC in the PLFE liposomes (1:500). The slope of F48O/F395 vs. pressure is positive between 16.9’C to 47.5 “C. At 47.5 ‘C, the slope is zero, and by 56.6 ‘C F48O/F395 starts to decrease with pressure. R drops from a slightly positive 0.0053 at 16.9”C to a value of -0.0053 at 56.6 “C. 1243
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Figure 2.
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2.0
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F480/F395 for PyrPCin PLFE liposomesasa function of temperatureand pressure. A.(O)=16.9 ‘C;(O)= 26.9 “C; and (0) = 37.6 “C. B. A = 47.5 ‘C and (A) = 56.6.V. [PLFE]/[PyrPC] = 500/l.
Positive values of R are not observed for any other lipids. The zero R-value at 48 ‘C suggests that at this temperature PyrPC is virtually laterally immobile in the PLPE liposomes and starts gaining some lateral mobility only for T > 48 “C. The positive R-values observed for T c 48 ‘C are rather surprising. This anomaly may be interpreted within the context of “monomer pairs” (1X,19), which predicts that excimers of pyrene will form only if the pyrene rings of the neighboring monomer pairs are oriented in an appropriate direction, presumably in a parallel disposition. In the temperature range of 15 - 48 ‘C, PLFE liposomes appear to be very rigid. Pressure probably has little infhtence on the lateral mobility of PyrPC. However, in such a rigid environment, pressure may alter the mutual orientation of the pyrene rings between the neighboring pyrene monomer pairs, forcing the neighboring pyrene rings to align parallel to each other. As a result, the E/M increases with pressure, giving the positive anomalous R values. According to this explanation, both R = 0 and R > 0 can be considered as indicating that no significant lateral mobility exists for the probe. The R values of all the lipid systems examined are summarized in Figure 3. Branched methyl-groups in diphyPC significantly affect interchain packing and lipid dynamics due to steric effects (20-22). Stewart et al. (20) have previously shown that increased branching decreases the 1244
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(C)
Temperature dependence of R in various lip&omes. R is the change in F48O/F395 perkbar. PLFE (1:SOO); PLFE (1:X)); POPC (1:SOO); diphyPC (1:500); C(18):C(lO)PC (1:500); and DMPC (1:250). All F48O/F395 values were normalized to a PyrPCxnatrix lipid molar ratio of 1:500.(O)= PLFE (1:500),(e)= PLFE (1:50),(x)= DMPC, (m) = DiphyPC, (Cl) = POPC, and(A)= C(18):C(lO)PC.
membrane order. This strongly suggests that branching can be a way to maintain the membranes in a disordered state over a wide temperature, pressure, and pH range. Our results shown in Figure 3 indicate that the R values for diphyPC, while higher than for straight-chain lipids, are more negative than for PLFR, suggesting that the low lateral-mobility of PyrPC in PLFE liposomes cannot be wholly attributed to branched methyl groups. C( lS):C( lO)PC, which forms mixed interdigitated structures below T,,, (18.8 “C) and partially interdigitated structures above T,,, was used to test whether the tightly packed, interdigitated state would present a similar matrix environment as PLFE. Also, in the mixed interdigitated form, one of the two acyl chains interpenetrates across the entire membrane, resembling the cross-linked structure of PLFEZ. However, it is seen in Figure 3 that PyrPC in C( 1S):C( 10)PC exhibits considerable lateral mobility. Thus, it is also unlikely that the low lateral mobility of PyrPC in PLFE liposomes can be attributed to monomolecular packing alone. While lateral mobility of PyrPC in PLFE liposomes is affected by the probe:lipid ratio (Figure 3), previous studies suggested that PyrPC does not form segregated domains in liposomes in the probe concentrations examined (0.2 mol% - 2 mol%) (6,24,25). Also, epifluorescence microscopy observations of the fluorophore distribution of N-lissamine-rhodamine B sulphonyl diacyl phosphatidylethanolamine (1 mol%) in PLFE do not show probe phase-separation (private communication with Dr. A. Gliozzi). If this situation holds true for PyrPC in PLFE liposomes, then the decrease in R as probe concentration increases can be ascribed to probe-induced increase in membrane perturbation, which increases the lateral mobility of PyrPC. When the temperature for which R = 0 is extrapolated to the case of zero probe-concentration, then R = 0 at about 58 ‘C for pure PLFE lipids, which is close to the minimum gxowth temperature of S. acidoculdurius . This implies that PLPE liposomes begin to gain fluidity at T > 58 OCand may be taken to explain partially why S. acidocaldarius is adapted to living at high temperatures, 1245
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RESEARCH COMMUNICATIONS *
Acknowledgments This work was supported by the NSF-RIM1 (RII-9014052). This work was done during the tenure of an Established Investigatorship (to P. L.-G. C.) from the American Heart Association and CIBA-GEIGY. E.L.C. would like to acknowledge support from the Office of Naval Research, through the Core Program at Naval Research Laboratory.
References 1. 2 i: 5. 6. 7. 8. 9. 10. 11 12. 13. 14. 15. 16. 17. 18. :;: 2 23: 24. 25.
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