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Lateral phase separations in lipid lamellar phases at low hydration
ABSTRACTS.
25th ANNUAL
We compared the ability of various amino acids to protect small unilamellar vesicles against damage during freeze/thaw. Liposomes were composed of 75% palmitoyloleoyl phosphatidylcholine and 25% phosphatidylserine. Damage to liposomes frozen in liquid nitrogen and thawed at 20°C was assessed by resonance energy transfer. Cryoprotection by numerous amino acids was compared in the presence and absence of 350 m&f NaCl. The majority of amino acids with hydrocarbon side chains increased membrane damage during freeze/thaw regardless of the presence of salt. However, amino acids with hydrocarbon side chains less than three carbons long, e.g., glycine, alanine, and 2-aminobutyric acid, were cryoprotective only in the presence of salt. It is suggested that NaCl selectively increases the solubility of certain amino acids, allowing these compounds to act as cryoprotectants. In contrast, amino acids with side chains containing charged amine groups were cryoprotective regardless of the presence of salt. The degree of charge on the second amine group is shown to be important for protection by these molecules. Evidence is presented that suggests an interaction between the positively charged, second amine group of the amino acid, and the negatively charged phospholipid headgroup. 95. Dry Dipalmitoylphosphatidylcholine and Trehalose Revisited. L. M. CROWEAND J. H.
CROWE(Department of Zoology, University of California, Davis, California 95616). We have reinvestigated the interactions of the disaccharide, trehalose, with bilayers of dipalmitoylphosphatidylcholine (DPPC) in order to rectify some of the unavoidable experimental defects of the original study (Crowe et al., Science, 223 (1984). Specifically, our mixtures of lipid and carbohydrate have been formed from an aqueous rather than organic medium and the mixtures have been kept as dry as possible at all stages to eliminate the effects of slight and unquantified hydration. Unilamellar vesicles were formed from DPPC by sonication (av. diameter, 25 nm) or by extrusion (av. diameter, 90 nm) and then either lyophilized (gel phase) or dried over anhydrous CaSO, at 60°C (liquid crystalline phase), followed by exposure to a vacuum. The effects of trehalose on the gel to liquid crystalline transition were even more striking than in our previous study: at a 1:1 mass ratio, trehalose decrease T,,, of dry DPPC approximately 80°C (from 105 to 24°C. The effect of trehalose on T,,, depended on the amount of trehalose present and whether the sample was dried from above or below T,. The effect of storage at low temperatures was also studied: samples stored at 4-6’C for about 4 weeks showed a reversion to a higher transition temperature; i.e., they packed as if they had been dried in gel phase. A phase diagram for dry DPPC/trehalose is presented. Results indicate that the PC headgroups require the expanded liquid crys-
541
MEETING
talline spacing for maximum interaction with trehalose, as previously suggested by Gaber et al. (Biophys. J. 49,435a (1986)) for DMPC. (Supported by Grant DMB 85-18194from NSF.) 96. Lateral Phase Separations Phases at Low Hydration.
in Lipid Lamellar G. BRYANT AND J.
WOLFE (Department of Biophysics, University of New South Wales, Kensington, Australia). Lateral phase separations have been produced in the membranes of protoplasts by freeze-induced dehydration and by osmotic dehydration at 0°C (1). The inverse hexagonal phase was also observed and correlated with lack of osmotic response in the thaw of a freeze-thaw cycle. At the very low chemical potential of water which is produced by freezing, interbilayer interactions are dominated by hydration forces which have been measured by Lis et al. (2) This short-range repulsion can produce large lateral stresses in the membranes (3). We present an analysis of the phase behaviour of mixed lipid lamellar phases. For lipids with sufftciently different hydration forces, and at low hydration, our calculations predict separation into two fluid lamellar phases with different spacings and different lateral stresses. We present phase diagrams for mixtures of neutral lipids with no enthalpy of mixing. (A similar calculation explains the inhomogenous distribution of membrane proteins.) This has implications for the freezing injury due to dehydration. Membrane containing mixtures of strongly repelling lipids (e.g., phosphatidylcholine) and weakly repelling lipids (e.g., glucocerebrosides) should form local, highly concentrated domains of the weakly repelling species in the membranes of dehydrated cells. These domains would also sequester the lipids most likely to form the inverse hexagonal phase. REFERENCES
1. Gordon-Kamm, W. J., and Steponkus, P. L. Lamellar-to-hexagonal II phase transitions in the plasma membrane of isolated protoplasts after freeze-induced dehydration. Proc. Natl. Acad. Sci. USA 81, 6373-6377 (1984).
2. Lis, L. J., McAlister, M., Rand, R. P., and Parsegian, V. A. Interactions between neutral phospholipid bilayer membranes. Biophys. J. 37, 657-666. (1982). 3. Wolfe, J. Lateral stresses in membranes at low water potential. Aust. J. Plant Physiol. 14, 311318 (1987). SESSION XII-CRYOPRESERVATION AND TRANSPLANTATION OF ISLETS OF LANGERHANS 97. Islet Bankingfor
Transplantation
in Diabetes Mel-
25th ANNUAL
We compared the ability of various amino acids to protect small unilamellar vesicles against damage during freeze/thaw. Liposomes were composed of 75% palmitoyloleoyl phosphatidylcholine and 25% phosphatidylserine. Damage to liposomes frozen in liquid nitrogen and thawed at 20°C was assessed by resonance energy transfer. Cryoprotection by numerous amino acids was compared in the presence and absence of 350 m&f NaCl. The majority of amino acids with hydrocarbon side chains increased membrane damage during freeze/thaw regardless of the presence of salt. However, amino acids with hydrocarbon side chains less than three carbons long, e.g., glycine, alanine, and 2-aminobutyric acid, were cryoprotective only in the presence of salt. It is suggested that NaCl selectively increases the solubility of certain amino acids, allowing these compounds to act as cryoprotectants. In contrast, amino acids with side chains containing charged amine groups were cryoprotective regardless of the presence of salt. The degree of charge on the second amine group is shown to be important for protection by these molecules. Evidence is presented that suggests an interaction between the positively charged, second amine group of the amino acid, and the negatively charged phospholipid headgroup. 95. Dry Dipalmitoylphosphatidylcholine and Trehalose Revisited. L. M. CROWEAND J. H.
CROWE(Department of Zoology, University of California, Davis, California 95616). We have reinvestigated the interactions of the disaccharide, trehalose, with bilayers of dipalmitoylphosphatidylcholine (DPPC) in order to rectify some of the unavoidable experimental defects of the original study (Crowe et al., Science, 223 (1984). Specifically, our mixtures of lipid and carbohydrate have been formed from an aqueous rather than organic medium and the mixtures have been kept as dry as possible at all stages to eliminate the effects of slight and unquantified hydration. Unilamellar vesicles were formed from DPPC by sonication (av. diameter, 25 nm) or by extrusion (av. diameter, 90 nm) and then either lyophilized (gel phase) or dried over anhydrous CaSO, at 60°C (liquid crystalline phase), followed by exposure to a vacuum. The effects of trehalose on the gel to liquid crystalline transition were even more striking than in our previous study: at a 1:1 mass ratio, trehalose decrease T,,, of dry DPPC approximately 80°C (from 105 to 24°C. The effect of trehalose on T,,, depended on the amount of trehalose present and whether the sample was dried from above or below T,. The effect of storage at low temperatures was also studied: samples stored at 4-6’C for about 4 weeks showed a reversion to a higher transition temperature; i.e., they packed as if they had been dried in gel phase. A phase diagram for dry DPPC/trehalose is presented. Results indicate that the PC headgroups require the expanded liquid crys-
541
MEETING
talline spacing for maximum interaction with trehalose, as previously suggested by Gaber et al. (Biophys. J. 49,435a (1986)) for DMPC. (Supported by Grant DMB 85-18194from NSF.) 96. Lateral Phase Separations Phases at Low Hydration.
in Lipid Lamellar G. BRYANT AND J.
WOLFE (Department of Biophysics, University of New South Wales, Kensington, Australia). Lateral phase separations have been produced in the membranes of protoplasts by freeze-induced dehydration and by osmotic dehydration at 0°C (1). The inverse hexagonal phase was also observed and correlated with lack of osmotic response in the thaw of a freeze-thaw cycle. At the very low chemical potential of water which is produced by freezing, interbilayer interactions are dominated by hydration forces which have been measured by Lis et al. (2) This short-range repulsion can produce large lateral stresses in the membranes (3). We present an analysis of the phase behaviour of mixed lipid lamellar phases. For lipids with sufftciently different hydration forces, and at low hydration, our calculations predict separation into two fluid lamellar phases with different spacings and different lateral stresses. We present phase diagrams for mixtures of neutral lipids with no enthalpy of mixing. (A similar calculation explains the inhomogenous distribution of membrane proteins.) This has implications for the freezing injury due to dehydration. Membrane containing mixtures of strongly repelling lipids (e.g., phosphatidylcholine) and weakly repelling lipids (e.g., glucocerebrosides) should form local, highly concentrated domains of the weakly repelling species in the membranes of dehydrated cells. These domains would also sequester the lipids most likely to form the inverse hexagonal phase. REFERENCES
1. Gordon-Kamm, W. J., and Steponkus, P. L. Lamellar-to-hexagonal II phase transitions in the plasma membrane of isolated protoplasts after freeze-induced dehydration. Proc. Natl. Acad. Sci. USA 81, 6373-6377 (1984).
2. Lis, L. J., McAlister, M., Rand, R. P., and Parsegian, V. A. Interactions between neutral phospholipid bilayer membranes. Biophys. J. 37, 657-666. (1982). 3. Wolfe, J. Lateral stresses in membranes at low water potential. Aust. J. Plant Physiol. 14, 311318 (1987). SESSION XII-CRYOPRESERVATION AND TRANSPLANTATION OF ISLETS OF LANGERHANS 97. Islet Bankingfor
Transplantation
in Diabetes Mel-