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Nuclear magnetic resonance studies of amphiphile hydration
ARCHNES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 187, No. 1, April 15, pp. 197-200, 1978
Nuclear
Magnetic
Effects
of the Gel-to-Liquid
RONALD
P. TAYLOR,’
Department
of Biochemistry,
Resonance Crystalline
Studies
Phase Transition Lecithin
CHING-HSIEN HUANG, JIN K. CHUN University
of Amphiphile
of Virginia,
Hydration’
on the Hydration
ANTHONY
School of Medicine,
of Dioleoyl
V. BROCCOLI,
Charlottesville,
Virginia
AND
22901
Received October 17, 1977; revised December 27, 1977 The hydration of dioleoyl lecithin (DOL) and dimyristoyl lecithin (DML) has been measured as a function of temperature between -15 and -30°C. using low-temperature proton magnetic resonance. The hydration of DOL is considerably higher than that of DML. We detect 9 mol of unfrozen water/m01 of phospholipid at -25°C (our “standard” temperature) for DOL, and only 6 mol of water/m01 of phospholipid for DML. The gel-toliquid crystalline phase transition in DOL centered at ca. -19°C is manifested by a 70% increase in hydration for both vesicles and dispersions. Preparations of either DML vesicles or vesicles of DOL which contain 33 mol% cholesterol would not be expected to undergo this phase transition, and the hydration increase observed for these preparations in the same range of temperature is less than 20%.
There is increasing interest in understanding in detail the nature of the water which interacts with cell membranes, because it is suspected that this water may play a significant role in modulating both the structural and functional properties of the cell membrane (l-5). It is known, for example, that the gel-to-liquid crystalline phase transition in phospholipid model membranes (generally characterized as a partial “melting” of the fatty acid chains in the bilayer) depends not only upon the nature of the phospholipid head group and its fatty acid side chains, but also upon the state of hydration of the phospholipid (6-8). Reciprocally, therefore, it might be expected that physical changes which occur in the bilayer would in fact be transmitted to the surrounding water of hydration as ‘This was supported by a Grant-in-Aid from the American Heart Association and by funds contributed by the Virginia Heart Association. This paper is No. 3; see Refs. (11) and (12) for the lirst two papers in this series. 2 Research Career Development Awardee of the National Institutes of Health, No. AI-000062-03.
well. There is some recent evidence wh@h indicates that this does occur (4, 5, 9). We have been using the low-temperature proton magnetic resonance technique of Kuntz et al. (10) to examine the nature of bound (“unfreezable”) water in frozen aqueous solutions of phospholipids (11,12), and in this paper we report that the gel-to-liquid crystalline transition centered at ca. -19OC in dioleoyl lecithin (DOL;3 1,2-dioleoyl-sn-glycero-3-phosphorylcholine) is paralleled by an approximately 70% increase in the water of hydration of the phospholipid. MATERIALS
AND
METHODS
1,2-Dimyristoyl phosphatidylcholine (DML, 1,2-d& myristoyl lecithin) was obtained from Sigma Chemical Co. DOL was synthesised following the procedures of Robles and Van Den Berg (13), and gave one spot on thin-layer chromatography using a solvent system consisting of chloroform-methanol-water (65:25:4, v/v/v). Vesicles of DML were prepared by sonication of an aqueous dispersion of the material at ca. 3O”C, followed by centrifugation to “size” the material (12). ’ Abbreviations used: DOL, 1,2-dioleoyl-sn-glycero3-phosphorylcholine; DML, 1,2-dimyristoyl lecithin; PC, phosphatidylcholine.
197 ooO3-9861/78/1871-0153~.00/0 Copyright
0 1978 by Academic
Press,
1,~.
All rights of reproduction in any form reserved
198
TAYLOR
The other materials used in the experiments, the methods of preparation of vesicles and dispersions, and the low-temperature hydration measurements have all been described (10-12). RESULTS
AND
DISCUSSION
On the influence of the acyl chains on hydration. It is an interesting question whether or not the introduction of a double bond in a phospholipid system of defined acyl chains (e.g., in DOL) causes a net increase or a net decrease in the water of hydration compared to a saturated system (e.g., DML). The vapor-phase adsorption studies of Jendrasiak and Hasty (14) suggest that the introduction of the double bond increases the water of hydration of phosphatidylcholine. Recent 13Cmagnetic resonance studies of the spin-lattice relaxation times ( 2’1)of the choline atoms in such systems indicate that in DOL there is a significant increase in the 2’1of the carbon atoms of the choline moiety relative to the saturated species (15). This implies that there is increased molecular motion in the choline atoms, but, as has been pointed out by Thompson and Huang (16), these results can in fact be interpreted in terms of either an increase or a decrease in hydration of the molecules. The increased motion could mean that there is a decrease in the intermolecular ionic attraction between the positively charged choline head group and the neighboring phosphate. This would allow for an increase in hydration, because it would have the effect of increasing the size of the potential hydration cavity surrounding the polar head groups as they moved further apart due to a decreasing interaction. Alternatively, the increased motion of the choline atoms could mean that their hydrodynamic mass decreased due to a dehydration mechanism relative to the saturated species. Our results strongly suggest that the introduction of the double bond causes a hydration increase. The integrated area of the proton magnetic resonance signal of unfrozen water (which we take as equivalent to the water of hydration) indicates that, at -25”C, the hydration of DOL (9 mol of water/m01 of phospholipid) is considerably greater than that of egg PC (6.5 mol/mol) or DML (6.0 mol/mol).
ET AL.
On the influence of the gel + liquid crystalline transition on hydration. The net hydration of DOL shows a marked temperature dependence in the region of -15 to -25°C (Fig. 1): The results suggest that there is an approximately 70% increase in hydration for both DOL vesicles and dispersions within this interval The center of this “transition” appears to be at ca. -19”C, which is rather close to the published value for the temperature of the gel-to-liquid crystalline phase transition of DOL, which is believed to be at about -20 to -22°C (7, 17, 18), although one recent report (19) suggests that the transition temperature is Only -140c. As a check on our hydration experiment, we examined two “control” samples which should not have any detectable phase transitions in the region of temperature under investigation. The corresponding transition for DML is at ca. +23”C (7) and, based on a wide range of experiments (7, 20-22), we can predict that the presence of 33 mol% cholesterol in DOL vesicles should, in fact, abolish the gel-to-liquid crystalhne transition in this system. Consistent with these predictions, we find that DML vesicles and 33 mol% cholesterol-DO1 vesicles exhibited comparatively small changes in hydration in the same region of temperature which was examined for DOL (Fig. 1).5 We conclude from these experiments that the increase in hydration observed for DOL preparations can be identified with the gelto-liquid crystalline transition in the bilayer of these systems. Although the principal changes in this melting transition are believed to occur in the hydrocarbon region “The errors in hydration were generally +lO% or less (at -25°C). However, there was some variability in the magnitude of the net hydration increase observed for DOL at -15°C (see Fig. l), and for this reason all of the data were displayed to represent accurately the experimental uncertainties at higher temperature. Further changes in the region between -10 and -15°C were small (ca. 10%) for all of the systems examined. Hysteresis effects were small (ca. 10% or less in normalized hydration). 5 Linewidth changes in these systems were not partitularly dramatic in the range of temperature under investigation. At -25°C the linewidth of the hydration signal for both DOL and DML was ca. 150 Hz (*25%), and generally decreased to ca. 10()-130 Hx at -15°C for both samples.
NMR
STUDIES
OF AMPHIPHILE
199
HYDRATION
2.0 r
I
1.6
I
I
I
I
-15
-20
-25
-30
TEMPERATURE,“C
FIG. I. Hydration
of phospholipids as a function of temperature. Hydration values have been normalized to 1.0 at -25”C, which corresponds to about 9.0 mol of water/m01 of DOL, 6.0 mol of water/m01 of DML, and about 12.0 mol of water/m01 of DOL for DOL vesicles containing 33 mol% cholesterol. The typical concentration of phospholipid in these systems (for both vesicles and dispersions) was about 40 mM in lipid phosphorus. Samples were prepared in unbuffered, distilled, deionized water. The pH of the solutions was generally between 5 and 7. Points are as follows: (V) and (O), DOL dispersions (unsonicated), two independent experiments (separate runs); (M), (A), and (+), DOL vesicles (sonicated and sized), three independent experiments; (0) and (Cl), DML vesicles, two independent experiments; (0) and (A), DOL vesicles containing 33 mol% cholesterol, two independent experiments. The solid line was drawn to emphasize the behavior of the systems which do not undergo a phase transition in the temperature range under investigation. The dashed line was drawn through the averaged values for those systems expected to show the thermal transition.
of the molecule (6, 7, 23), it is reasonable, based on a variety of model (24-28) studies, that the hydration changes we observe are, in fact, associated with the polar head groups, which would be expected to contain the principal hydration sites for the mole-
cule. Recent magnetic resonance studies indicate that the gel-to-liquid crystalline phase transition in many phospholipids does increase the mobility of the atoms in the head group region of the molecules, as well as that of the whole molecule (6,
200
TAYLOR
29-33). It is likely that the increased mobility in the polar head group region of the molecules occurs concomitantly with an increase in the size of the potential hydration cavity surrounding these groups, because, in effect, they can “sweep out” a greater volume and perturb more water. The marked increase in hydration that we observed for DOL above the phase transition is certainly consistent with this hypothesis. We note that other experiments have provided evidence for changes in the nature of the water of hydration of phosphatidylcholine (PC) above its phase transition (1,4,5, 9), but we believe that this experiment constitutes the first direct evidence demonstrating that the transition actually causes a net increase in hydration. Whether this thermally induced increase in hydration which we observed is of importance in biological systems obviously remains open to question. We mentioned that the functional properties of a membrane may depend upon its surrounding water of hydration (l-5). It is known that other parameters, such as pH, specific metal ions, and drugs, can also perturb the gel-to-liquid crystalline transition in phospholipids (7, 29, 30, 33, 34) and, in so doing, affect a number of physical properties of these systems, including their permeability. Thus, it will be most interesting to see if, by manipulating these variables to cause the transition in the appropriate system, we can in fact detect the expected hydration increase at a fixed temperature. ACKNOWLEDGMENTS We thank Dr. Peter Russell and Dr. Tsin Wei for helpful discussions. REFERENCES 1. KEITH, A. D., SNIPES, W., AND CHAPMAN, D. (1977) Biochemistry 7,634. 2. INGLEFIELD, P. T., LINDBLOM, K. A., AND GOTTLIEB, A. M. (1976) Biochem. Biophys. Acta 419, 196. 3. NEWMAN, G. C., AND HUANG, C. (1975) Biochemistry 14, 3363. 4. PRESTEGARD, J. H., AND WILKINSON, A. (1974)
Biochem. Biophys. Acta 345,439. 5. FINER, E. G., AND DARKE, A. (1974) Chem. Phys. Lipids 12, 1. 6. LADBROOKE, B. C., AND CHAPMAN, D. (1969)
Chem. Phys. Lipids 3,304. 7. CHAPMAN, D. (1973) in Biological
Membranes
ET AL.
8. 9. 10. 11. 12.
13. 14.
(Chapman, D., and WaIlach, D. F. H., eds.), Vol. 2, Chap., 2 Academic Press, New York. JANIAK, M. J., SMALL, D. M., AND SHIPLEY, G. G. 15,4575. (1976) Biochemdy SALSBURY, N. J., DARKE, A., AND CHAPMAN, D. (1972) Chem. Phys Lipids 8,142. KUNTZ, I. D., BRASSFIELD, T. S., LAW, G. D., AND PURCELL, A. V. (1969) Science 163,1329. TAYLOR, R. P. (1976) Arch. B&hem. Biophys. 173,596. TAYLOR, R. P., HUANG, C., BROCCOLI, A. V., AND LEAKE, L. (1977) Arch. Biochem. Biophys. 183, 83. ROBLES, E. C., AND VAN DEN BERG, D. (1969) B&hem. Biaphys. Acta 187,520. JENDRASIAK, G. L., AND HASTY, J. H. (1974)
Biochim. Biophys. Acta 337,79. 15. LEVINE, Y. K., BIRDSALL, N. J. M., LEE, A. G., AND METCALFJX, J. C. (1972) Biochemistry 11, 1416. 16. THOMPSON, T. E., AND HUANG, C. (1978) in The Physiological Basis for Disorders of Biomembranes (Andrioli, T. E., Hoffman, J. F., and Fanestil, D. D., eds.), Plenum Press, New York (in press). 17. BARTON, P. G., AND GUNSTONE, F. D. (1975) J.
Biol. Chem 250.4470. 18. DE KRUIJFF, B., CULLIS, P. R., AND RADDA, G. K. (1975) B&him. Biophys. Acta 406,6. 19. VAN DIJCK, P. W. M., DE KRUIJFF, B., VAN DEENAN, L. L. M., DEGIER, J., AND DEMEL, R. A. (1976) B&hem. Biophys. Acta 455,576. 20. PHILLIPS, M. C., AND FINER, E. G. (1974) B&him.
Biophys. Acta 356,199. 21. ENGLEMAN, D. M., AND ROTHMAN, J. E. (1972) J.
Biol. Chem. 247,3694. 22. HINZ, H. J., AND STUTEVANT, J. M. (1972) J. Biol.
Chem. 247,3697. 23. LEE, A. G. (1977) B&him. Biophys. Acta 472, 237. 24. KUNTZ, I. D. (1971) J. Amer. Chem. Sot. 93,514. 25. FRINGELI, U. P., AND G~NTHARD, H. H. (1976) B&him. Biophys. Acta 450, 101. 26. WELLS, M. A. (1974) Biochemistry 13,4937. 27. PHILLIPS, M. C., FINER, E. G., AND HAUSER, H. (1972) Biochim. Biophys. Acta 290,397. 28. DAVENPORT, J. B., AND FISHER, L. R. (1975) Chem. Phys. Lipids 14,275. 29. LEE, A. G. (1975) Progr. Biophys. Mol. Bid. 29, 3. 30. CULLIS, P. R., AND DEKRUYFF, B. (1976) B&him.
Biophys. Acta 436,523. 31. SEELIG, J., AND GALLY, H.-V. (1976) Biochemistry 15,5199. 32. KOHLER, S. J., AND KLEIN, M. P. (1977) Biochemistry 16,519. 33. CHAPMAN, D. (1975) Quart Reu. Biophys. 8,185. 34. CHAPMAN, D., PEEL, W. E., KINGSTON, B., AND LILLEY, T. H. (1977) Biochim. Biophys. Acta 464,260.
Nuclear
Magnetic
Effects
of the Gel-to-Liquid
RONALD
P. TAYLOR,’
Department
of Biochemistry,
Resonance Crystalline
Studies
Phase Transition Lecithin
CHING-HSIEN HUANG, JIN K. CHUN University
of Amphiphile
of Virginia,
Hydration’
on the Hydration
ANTHONY
School of Medicine,
of Dioleoyl
V. BROCCOLI,
Charlottesville,
Virginia
AND
22901
Received October 17, 1977; revised December 27, 1977 The hydration of dioleoyl lecithin (DOL) and dimyristoyl lecithin (DML) has been measured as a function of temperature between -15 and -30°C. using low-temperature proton magnetic resonance. The hydration of DOL is considerably higher than that of DML. We detect 9 mol of unfrozen water/m01 of phospholipid at -25°C (our “standard” temperature) for DOL, and only 6 mol of water/m01 of phospholipid for DML. The gel-toliquid crystalline phase transition in DOL centered at ca. -19°C is manifested by a 70% increase in hydration for both vesicles and dispersions. Preparations of either DML vesicles or vesicles of DOL which contain 33 mol% cholesterol would not be expected to undergo this phase transition, and the hydration increase observed for these preparations in the same range of temperature is less than 20%.
There is increasing interest in understanding in detail the nature of the water which interacts with cell membranes, because it is suspected that this water may play a significant role in modulating both the structural and functional properties of the cell membrane (l-5). It is known, for example, that the gel-to-liquid crystalline phase transition in phospholipid model membranes (generally characterized as a partial “melting” of the fatty acid chains in the bilayer) depends not only upon the nature of the phospholipid head group and its fatty acid side chains, but also upon the state of hydration of the phospholipid (6-8). Reciprocally, therefore, it might be expected that physical changes which occur in the bilayer would in fact be transmitted to the surrounding water of hydration as ‘This was supported by a Grant-in-Aid from the American Heart Association and by funds contributed by the Virginia Heart Association. This paper is No. 3; see Refs. (11) and (12) for the lirst two papers in this series. 2 Research Career Development Awardee of the National Institutes of Health, No. AI-000062-03.
well. There is some recent evidence wh@h indicates that this does occur (4, 5, 9). We have been using the low-temperature proton magnetic resonance technique of Kuntz et al. (10) to examine the nature of bound (“unfreezable”) water in frozen aqueous solutions of phospholipids (11,12), and in this paper we report that the gel-to-liquid crystalline transition centered at ca. -19OC in dioleoyl lecithin (DOL;3 1,2-dioleoyl-sn-glycero-3-phosphorylcholine) is paralleled by an approximately 70% increase in the water of hydration of the phospholipid. MATERIALS
AND
METHODS
1,2-Dimyristoyl phosphatidylcholine (DML, 1,2-d& myristoyl lecithin) was obtained from Sigma Chemical Co. DOL was synthesised following the procedures of Robles and Van Den Berg (13), and gave one spot on thin-layer chromatography using a solvent system consisting of chloroform-methanol-water (65:25:4, v/v/v). Vesicles of DML were prepared by sonication of an aqueous dispersion of the material at ca. 3O”C, followed by centrifugation to “size” the material (12). ’ Abbreviations used: DOL, 1,2-dioleoyl-sn-glycero3-phosphorylcholine; DML, 1,2-dimyristoyl lecithin; PC, phosphatidylcholine.
197 ooO3-9861/78/1871-0153~.00/0 Copyright
0 1978 by Academic
Press,
1,~.
All rights of reproduction in any form reserved
198
TAYLOR
The other materials used in the experiments, the methods of preparation of vesicles and dispersions, and the low-temperature hydration measurements have all been described (10-12). RESULTS
AND
DISCUSSION
On the influence of the acyl chains on hydration. It is an interesting question whether or not the introduction of a double bond in a phospholipid system of defined acyl chains (e.g., in DOL) causes a net increase or a net decrease in the water of hydration compared to a saturated system (e.g., DML). The vapor-phase adsorption studies of Jendrasiak and Hasty (14) suggest that the introduction of the double bond increases the water of hydration of phosphatidylcholine. Recent 13Cmagnetic resonance studies of the spin-lattice relaxation times ( 2’1)of the choline atoms in such systems indicate that in DOL there is a significant increase in the 2’1of the carbon atoms of the choline moiety relative to the saturated species (15). This implies that there is increased molecular motion in the choline atoms, but, as has been pointed out by Thompson and Huang (16), these results can in fact be interpreted in terms of either an increase or a decrease in hydration of the molecules. The increased motion could mean that there is a decrease in the intermolecular ionic attraction between the positively charged choline head group and the neighboring phosphate. This would allow for an increase in hydration, because it would have the effect of increasing the size of the potential hydration cavity surrounding the polar head groups as they moved further apart due to a decreasing interaction. Alternatively, the increased motion of the choline atoms could mean that their hydrodynamic mass decreased due to a dehydration mechanism relative to the saturated species. Our results strongly suggest that the introduction of the double bond causes a hydration increase. The integrated area of the proton magnetic resonance signal of unfrozen water (which we take as equivalent to the water of hydration) indicates that, at -25”C, the hydration of DOL (9 mol of water/m01 of phospholipid) is considerably greater than that of egg PC (6.5 mol/mol) or DML (6.0 mol/mol).
ET AL.
On the influence of the gel + liquid crystalline transition on hydration. The net hydration of DOL shows a marked temperature dependence in the region of -15 to -25°C (Fig. 1): The results suggest that there is an approximately 70% increase in hydration for both DOL vesicles and dispersions within this interval The center of this “transition” appears to be at ca. -19”C, which is rather close to the published value for the temperature of the gel-to-liquid crystalline phase transition of DOL, which is believed to be at about -20 to -22°C (7, 17, 18), although one recent report (19) suggests that the transition temperature is Only -140c. As a check on our hydration experiment, we examined two “control” samples which should not have any detectable phase transitions in the region of temperature under investigation. The corresponding transition for DML is at ca. +23”C (7) and, based on a wide range of experiments (7, 20-22), we can predict that the presence of 33 mol% cholesterol in DOL vesicles should, in fact, abolish the gel-to-liquid crystalhne transition in this system. Consistent with these predictions, we find that DML vesicles and 33 mol% cholesterol-DO1 vesicles exhibited comparatively small changes in hydration in the same region of temperature which was examined for DOL (Fig. 1).5 We conclude from these experiments that the increase in hydration observed for DOL preparations can be identified with the gelto-liquid crystalline transition in the bilayer of these systems. Although the principal changes in this melting transition are believed to occur in the hydrocarbon region “The errors in hydration were generally +lO% or less (at -25°C). However, there was some variability in the magnitude of the net hydration increase observed for DOL at -15°C (see Fig. l), and for this reason all of the data were displayed to represent accurately the experimental uncertainties at higher temperature. Further changes in the region between -10 and -15°C were small (ca. 10%) for all of the systems examined. Hysteresis effects were small (ca. 10% or less in normalized hydration). 5 Linewidth changes in these systems were not partitularly dramatic in the range of temperature under investigation. At -25°C the linewidth of the hydration signal for both DOL and DML was ca. 150 Hz (*25%), and generally decreased to ca. 10()-130 Hx at -15°C for both samples.
NMR
STUDIES
OF AMPHIPHILE
199
HYDRATION
2.0 r
I
1.6
I
I
I
I
-15
-20
-25
-30
TEMPERATURE,“C
FIG. I. Hydration
of phospholipids as a function of temperature. Hydration values have been normalized to 1.0 at -25”C, which corresponds to about 9.0 mol of water/m01 of DOL, 6.0 mol of water/m01 of DML, and about 12.0 mol of water/m01 of DOL for DOL vesicles containing 33 mol% cholesterol. The typical concentration of phospholipid in these systems (for both vesicles and dispersions) was about 40 mM in lipid phosphorus. Samples were prepared in unbuffered, distilled, deionized water. The pH of the solutions was generally between 5 and 7. Points are as follows: (V) and (O), DOL dispersions (unsonicated), two independent experiments (separate runs); (M), (A), and (+), DOL vesicles (sonicated and sized), three independent experiments; (0) and (Cl), DML vesicles, two independent experiments; (0) and (A), DOL vesicles containing 33 mol% cholesterol, two independent experiments. The solid line was drawn to emphasize the behavior of the systems which do not undergo a phase transition in the temperature range under investigation. The dashed line was drawn through the averaged values for those systems expected to show the thermal transition.
of the molecule (6, 7, 23), it is reasonable, based on a variety of model (24-28) studies, that the hydration changes we observe are, in fact, associated with the polar head groups, which would be expected to contain the principal hydration sites for the mole-
cule. Recent magnetic resonance studies indicate that the gel-to-liquid crystalline phase transition in many phospholipids does increase the mobility of the atoms in the head group region of the molecules, as well as that of the whole molecule (6,
200
TAYLOR
29-33). It is likely that the increased mobility in the polar head group region of the molecules occurs concomitantly with an increase in the size of the potential hydration cavity surrounding these groups, because, in effect, they can “sweep out” a greater volume and perturb more water. The marked increase in hydration that we observed for DOL above the phase transition is certainly consistent with this hypothesis. We note that other experiments have provided evidence for changes in the nature of the water of hydration of phosphatidylcholine (PC) above its phase transition (1,4,5, 9), but we believe that this experiment constitutes the first direct evidence demonstrating that the transition actually causes a net increase in hydration. Whether this thermally induced increase in hydration which we observed is of importance in biological systems obviously remains open to question. We mentioned that the functional properties of a membrane may depend upon its surrounding water of hydration (l-5). It is known that other parameters, such as pH, specific metal ions, and drugs, can also perturb the gel-to-liquid crystalline transition in phospholipids (7, 29, 30, 33, 34) and, in so doing, affect a number of physical properties of these systems, including their permeability. Thus, it will be most interesting to see if, by manipulating these variables to cause the transition in the appropriate system, we can in fact detect the expected hydration increase at a fixed temperature. ACKNOWLEDGMENTS We thank Dr. Peter Russell and Dr. Tsin Wei for helpful discussions. REFERENCES 1. KEITH, A. D., SNIPES, W., AND CHAPMAN, D. (1977) Biochemistry 7,634. 2. INGLEFIELD, P. T., LINDBLOM, K. A., AND GOTTLIEB, A. M. (1976) Biochem. Biophys. Acta 419, 196. 3. NEWMAN, G. C., AND HUANG, C. (1975) Biochemistry 14, 3363. 4. PRESTEGARD, J. H., AND WILKINSON, A. (1974)
Biochem. Biophys. Acta 345,439. 5. FINER, E. G., AND DARKE, A. (1974) Chem. Phys. Lipids 12, 1. 6. LADBROOKE, B. C., AND CHAPMAN, D. (1969)
Chem. Phys. Lipids 3,304. 7. CHAPMAN, D. (1973) in Biological
Membranes
ET AL.
8. 9. 10. 11. 12.
13. 14.
(Chapman, D., and WaIlach, D. F. H., eds.), Vol. 2, Chap., 2 Academic Press, New York. JANIAK, M. J., SMALL, D. M., AND SHIPLEY, G. G. 15,4575. (1976) Biochemdy SALSBURY, N. J., DARKE, A., AND CHAPMAN, D. (1972) Chem. Phys Lipids 8,142. KUNTZ, I. D., BRASSFIELD, T. S., LAW, G. D., AND PURCELL, A. V. (1969) Science 163,1329. TAYLOR, R. P. (1976) Arch. B&hem. Biophys. 173,596. TAYLOR, R. P., HUANG, C., BROCCOLI, A. V., AND LEAKE, L. (1977) Arch. Biochem. Biophys. 183, 83. ROBLES, E. C., AND VAN DEN BERG, D. (1969) B&hem. Biaphys. Acta 187,520. JENDRASIAK, G. L., AND HASTY, J. H. (1974)
Biochim. Biophys. Acta 337,79. 15. LEVINE, Y. K., BIRDSALL, N. J. M., LEE, A. G., AND METCALFJX, J. C. (1972) Biochemistry 11, 1416. 16. THOMPSON, T. E., AND HUANG, C. (1978) in The Physiological Basis for Disorders of Biomembranes (Andrioli, T. E., Hoffman, J. F., and Fanestil, D. D., eds.), Plenum Press, New York (in press). 17. BARTON, P. G., AND GUNSTONE, F. D. (1975) J.
Biol. Chem 250.4470. 18. DE KRUIJFF, B., CULLIS, P. R., AND RADDA, G. K. (1975) B&him. Biophys. Acta 406,6. 19. VAN DIJCK, P. W. M., DE KRUIJFF, B., VAN DEENAN, L. L. M., DEGIER, J., AND DEMEL, R. A. (1976) B&hem. Biophys. Acta 455,576. 20. PHILLIPS, M. C., AND FINER, E. G. (1974) B&him.
Biophys. Acta 356,199. 21. ENGLEMAN, D. M., AND ROTHMAN, J. E. (1972) J.
Biol. Chem. 247,3694. 22. HINZ, H. J., AND STUTEVANT, J. M. (1972) J. Biol.
Chem. 247,3697. 23. LEE, A. G. (1977) B&him. Biophys. Acta 472, 237. 24. KUNTZ, I. D. (1971) J. Amer. Chem. Sot. 93,514. 25. FRINGELI, U. P., AND G~NTHARD, H. H. (1976) B&him. Biophys. Acta 450, 101. 26. WELLS, M. A. (1974) Biochemistry 13,4937. 27. PHILLIPS, M. C., FINER, E. G., AND HAUSER, H. (1972) Biochim. Biophys. Acta 290,397. 28. DAVENPORT, J. B., AND FISHER, L. R. (1975) Chem. Phys. Lipids 14,275. 29. LEE, A. G. (1975) Progr. Biophys. Mol. Bid. 29, 3. 30. CULLIS, P. R., AND DEKRUYFF, B. (1976) B&him.
Biophys. Acta 436,523. 31. SEELIG, J., AND GALLY, H.-V. (1976) Biochemistry 15,5199. 32. KOHLER, S. J., AND KLEIN, M. P. (1977) Biochemistry 16,519. 33. CHAPMAN, D. (1975) Quart Reu. Biophys. 8,185. 34. CHAPMAN, D., PEEL, W. E., KINGSTON, B., AND LILLEY, T. H. (1977) Biochim. Biophys. Acta 464,260.