tillant (Fraley et al. 1980). Generally 10% of the applied pBR322 DNA,. 10% of the DNase I, and 60-70% of the lipids were recovered in the liposomes. Liposome ...
J Mol Evol (1994) 39:560-568
Journal of Molecular Evolution © Springer-VerlagNewYorkInc. 1994
Transbilayer Diffusion of Divalent Cations into Liposomes Mediated by Lipidic Particles of Phosphatidate Isabel Baeza, 1 Carlos Wong, 1 Ricardo Mondrag6n, 2 Sirenia Gonzfilez, 3 Miguel Ibfifiez, 1 Norberto Farffin, 4 Carlos Argiiello s 1 Departamento de Bioquimica, Escuela Nacional de Ciencias Biol6gicas del IPN, 11340-Mrxico, D.F. 2 Departamento de Biologfa Celular, Centro de Investigaci6n y Estudios Avanzados del IPN, 07000 Mrxico, D.F. 3 Unidad de Microscopia Electr6nica, Centro de Investigaci6n y Estudios Avanzados del IPN, 07000 Mrxico, D.F. 4 Departamento de Qufmica, Centro de Investigaci6n y Estudios Avanzados del IPN, 07000 Mexico, D.F. 5 Departarnento de Patologia Experimental, Centro de Investigaci6n y Estudios Avanzados del IPN, Apartado Postal 14-740, 07000 Mrxico, D.F. Received: 22 December 1993 / Accepted: 12 May 1994
Abstract. Liposomes formed from egg-yolk phosphatidylcholine:egg-yolk phosphatidate (molar ratio 2:1) containing pBR322 DNA and DNase I were induced to form, with divalent cations, bilayer/nonbilayer phase transitions of phosphatidate which allowed cation diffusion into liposomes; then cation diffusion was measured by the activation of the hydrolysis of DNase I on DNA. The formation of phosphatidate transitions on liposomes was demonstrated by freeze-fracture and 3lp NMR, and a direct correlation between the formation of phosphatidate transitions and the transbilayer diffusion of cations was found: only Ca 2+ and Mn 2÷, which induce phase transitions, were able to penetrate liposomes and triggered the DNase I activity; in addition, Ca 2÷ at higher concentrations (10 mM) caused fusion of liposomes, whereas Mn 2÷ did not, suggesting that transitions induced by Mn ~+ participated only in the diffusion of this ion; furthermore, Mg 2+ neither formed phase transitions nor triggered the enzymatic activity. The liposomes studied represent more dynamic structures that can form phosphatidate structures involved in both (1) the interchange of divalent cations with the surroundings, thereby modulating encapsulated enzymes, and (2) the fusion of lipid vesicles probably implicated in the enrichment of liposomal content in the early Precambian Earth.
Correspondence to: C. Argtiello
Key words: Transbilayer transport - - Phase transitions - - Nonbilayer lipids - - Cone-shaped lipids - - Liposomes as a model of precellular systems
Introduction Amphiphilic lipids are the unique molecules that can self-assemble in a bilayer arrangement and form closed and self-sealing vesicles, or liposomes, which are wellknown models of primordial cell membranes (Hargreaves and Deamer 1978; Deamer and Or6 1980; Deamer and Barchfeld 1982). Liposomes offer a true barrier that effectively isolates microenvironments from the surroundings; however, the tightness of their lipid bilayers (Gruner 1987) leads to a permeability problem, which must be overcome if the lipid vesicles are to be considered as a good precellular model. A dynamic liposomal system capable of interchanging ions and polar molecules with the surrounding environment must be developed to further support the role of liposomes in the origin and evolution of life. For example, Nichols and Deamer (1976) showed that weak bases could be concentrated inside liposomes by the energy of pH gradients across their membranes, and Stillwell (1976) developed a prebiotic lipid model to transport amino acids into lipid vesicles. Since cone-shaped lipids undergo transitional changes
561
from bilayer to nonbilayer phase arrangements, such as H n and/or lipidic particles that could be involved in some biological functions--for example, fusion of membranes and transbilayer transport of ions (Verkleij 1984; Cullis et al. 1991)--we proposed that liposomes containing cone-shaped lipids could be more dynamic structures which can interchange ions with the surrounding environment. Effectively, we demonstrated Mn 2÷ diffusion into liposomes containing the cone-shaped lipid phosphatidate in their bilayers and DNA and DNase I inside of them. Mn z+ diffusion was monitored by the activation of the DNase I triggered by Mn 2+ when it penetrates liposomes (Baeza et al. 1990); however, the specific structural arrangement of phosphatidate in the lipid bilayers of liposomes was not analyzed. The purpose of this study was the detection by freeze-fracture and by 31p NMR of the nonbilayer phosphatidate phase transitions on liposomes and the analysis of their possible participation in the transbilayer movement of Mg 2÷, Ca 2+, and Mn 2÷. We found a direct correlation between the formation of phosphatidate phase transitions by Ca 2+ and Mn 2+ and their transbilayer diffusion into liposomes triggering the activity of liposomal DNase I; moreover, Ca 2÷ showed its fusogenic properties; in contrast, Mn 2÷ did not produce any fusion of liposomes, and Mg 2÷ did not show any of these features. These studies pointed out that the phosphatidate structures formed on liposomes by each one of the divalent cations could have different functions, such as: transbilayer diffusion of ions, which produced the activation of encapsulated enzymes, and fusion of lipid vesicles probably implicated in the enrichment of liposomal contents. The dynamic features of liposomes containing cone-shaped lipids improve their functional properties to be considered as an experimental model of precellular systems.
Liposome Preparation. Liposomes were prepared by the reversephase evaporation (REV) method (Szoka and Papahadjopoulos 1978). The method was used without reduced pressure (Baeza et al. 1990, 1992). Nine I.tmoles of lipids dissolved in 1 ml of diethyl ether were added to a 50-ml round-bottom flask, and 330 ~tl of 10 mM Tris-HCl, 1 mM NaCI, pH 7, containing 25 /.tg of pBR322 DNA, 0.1 /aCi of poly[5-3H]uridylic acid, and 9 × 10-2 Kunitz units of pancreatic DNase I were added. The resultant two-phase system was vortex-mixed and sonicated three times for 5 s in a bath-type sonicator (Lab Supply G112SOI). The ether was removed under nitrogen using rotary evaporation at 37°C, thereby producing the preparation of liposomes. These liposomes were separated from unencapsulated material by centrifugation at 200,000g for 30 min, and they were washed three times with the 10 mM Tris-HC1, 1 mM NaCI, pH 7 buffer solution (Fraley et al. 1980) and extruded through MF-Millipore membranes with 0.45-/am pores.
Materials and Methods
Liposome Leakage Assays. Vesicle leakage was measured by the method of Wilschut et al. (1980) and Gagng et al. (1985). Liposomes prepared by the REV method and containing 5(6)-carboxyfluorescein sodium salt (75 mM) were separated from the nonencapsulated fluorophore by gel filtration on Sephadex G-75 with 10 mM Tris-HCl, 1 mM NaC1, pH 7, as the elution buffer. Vesicle leakage during the incubation at 37°C with MgC12, CaCI2, or MnCI 2 among concentrations of 0.5 to 10 mM was measured by the carboxyfluorescein release with a Perking Elmer LS-5 spectrofluorometer. All fluorescence measurements were performed at an absorbance 0.2 OD units in 1.0-cm pathlength cuvettes. Carboxyfluorescein excitation was at 430 nm and its emission was detected at 530 nm. Complete release of carboxyfluorescein from vesicles was obtained by addition of Triton X-100 (0.1% v/v).
Materials. Egg-yolk phosphatidic acid (disodium salt) and egg-yolk phosphatidylcholine were purchased from Avanti Polar Lipids, Inc. (Birmingham, AL), and were used without further purification. Deoxyribonuclease I from bovine pancreas (DNase I) and 5(6)-carboxyfluorescein sodium salt were obtained from Sigma Chemical Co. (St. Louis, MO). Poly[5-3H]uridylic acid (20-72 Ci/mmol) was obtained from New England Nuclear (Boston, MA). Manganese chloride, calcium chloride, magnesium chloride, chloroform, diethyl ether, riffs, and EDTA were from Merck AG (Darmstadt, FRG). All other chemicals were of reagent quality.
DNA Preparation. The pBR322 plasmid DNA was obtained from Escherichia coli C600 by gentle phenol extractions (Birnboinm and Doly 1979). DNA was fractionated in a Sepharose 4B column for elimination of bacterial RNA; then, DNA was dialyzed against 10 mM Tris-HCl, 1 mM NaC1, pH 7, and kept at 5°C until used. The 260/280nm ratio of pBR322 DNA was 2.0.
DNA Concentration. DNA concentration was determined by Burton's method (Burton 1956) and by titration against a standard on an ethidum bromide-agarose plate (Maniatis et al. 1982).
Liposome Composition. Lipid concentration was calculated from quantitative determinations of phospholipids (Bottoger et al. 1961); the encapsulation of DNA and DNase I was determined by monitoring a 50-ktl aliquot of liposomes containing pBR322 DNA, DNase I, and poly[5-3H]uridylic acid for radioactivity in a Triton/toluene-based scintillant (Fraley et al. 1980). Generally 10% of the applied pBR322 DNA, 10% of the DNase I, and 60-70% of the lipids were recovered in the liposomes. Liposome Permeability to Divalent Cations. Liposome permeability was determined using the liposome transport system previously described (Baeza et al. 1990). Samples of the liposomes obtained from 9 ~tmoles of lipids and containing 2.5 ~tg of DNA and 9 × 10-3 Kunitz units of pancreatic DNase 1 were incubated with a 2 mM solution of either MgC12, CaC12, or MnC12, in 10 mM Tris-HC1, 1 mM NaC1, pH 7, at 37°C, several times. The reaction was stopped by adding 15 mM EDTA followed by an immediate extraction with phenol-chloroform 2: l (v/v). The aqueous-phase reaction products of DNase I, previously concentrated by lyophilization, were analyzed by electrophoresis in 1% agarose gels. Gels were stained with ethidium bromide and photographed with a Polaroid MP-4 camera and a Chromatovue transilluminator. Quantitative results were obtained by analyzing the gel negatives with the scanner Gelman DCD 16 (Baeza et al. 1987). As a control, 2.5 btg of pBR322 DNA was incubated with 9 × 10-3 Kunitz units of DNase I and with the cations; this sample was also extracted, lyophilized, and analyzed by electrophoresis as indicated above.
Freeze-Fracture Electron Microscopy. Samples of the liposomes studied by their permeability were processed for freeze-fracture; furthermore, we also analyzed liposomes without DNA and DNase 1 inside of them. Aliquots of liposomes obtained from 9 gmoles of eggyolk phosphatidylcholine or from egg-yolk phosphatidylcholine:eggyolk phosphatidate (2:1 molar ratio) in the 10 mM Tris-HC1, l mM NaC1, pH 7, buffer solution were incubated at 37°C during 30 rain in the absence or in the presence of 2 mM or 10 mM of MgCI2, or CaCI2, or MnCI 2. Then, liposomes were cooled down at 4°C during I h before
562 freeze-fracture. An aliquot of these liposomes was sandwiched between a pair of Balzers (Nashua, NH) copper support plates and frozen in Freon 22 cooled in liquid nitrogen (Singh et al. 1992). Glycerol at final concentration of 15% was added to prevent freeze damage of the lipid samples (Verkleij et al. 1982; Gaillard et al. 1991). Frozen samples were fractured in a double-replica device and replicated in a Balzers BAF 400D freeze-fracture unit equipped with electron beam guns, at a vacuum of 3 x 10.7 mbar, or better, with an incidence angle of 45 ° for platinum electrode and at -120°C. Replicas were floated off on distilled water and cleaned with concentrated chromic mixture; then, they were washed several times with distilled water. Finally, the replicas were picked up on 400-mesh copper grids covered with Formvar and viewed in JEOL 2000 EX electron microscope at 80 kV.
31p Nuclear Magnetic Resonance (NMR) Spectra. Samples of 100 gmoles of liposomes for 31p NMR spectra were withdrawn directly from the samples of vesicles without DNA and DNase I that were prepared for freeze-fracture; thus, they had the same history. These samples were incubated at 37°C in the absence or in the presence of divalent cations at 2 and 10 mM. 31p NMR spectra of lipid mixtures in 5-ram (OD) NMR tubes were obtained on a JEOL FX90Q multinuclear Fourier transform spectrofotometer operating at 36.23 MHz and at 37°C. A spectral width of 7.5 kHz was used with about 30,000 transients being accumulated with a 7-gs pulse and a 2.8-s pulse delay (Jones et al. 1992). Broad-band proton noise decoupling was used in all measurements and 2H20 was included in the lipid samples for spectrometer lock.
Results
Effect of Divalent Cations and Sonication on the Activity of Pancreatic DNase I Measuring the products of the hydrolytic activity of DNase I on DNA by electrophoresis in agarose gels, it was shown that the enzyme had a different activity in the presence of 2 mM of Mg 2+, Ca 2+, and Mn 2+ as cofactors. DNase I activity was higher in the presence of Mn 2+ (Fig. 1A, b,c) and lower with Mg 2+ (Fig. 1A, e,f), as had been previously shown by Maniatis et al. (1982); the lowest activity observed corresponded to Ca 2+ (Fig. 1A, h,i). This different effectivities of the cofactors were also observed when the cations were used at 10 mM. The demonstration that Ca z+ was also a cofactor of DNase I permitted us to determine the diffusion of the three cations into liposomes by measuring DNase I enzymatic activity. When the enzyme was sonicated separately from lipids, a notable decrease in its activity was found in the presence of Mn 2+ and Mg 2+ as compared with the nonsonicated enzyme (Fig. 1B, b,c and e,f), while in the presence of Ca 2+, a total loss in its activity was observed (Fig. 1B, h,i). Moreover, when the pBR322 DNA alone was sonicated as the enzyme, it had an extensive breakage (Fig. 1B, j).
Effect of Phosphatidate on the Permeability of Liposomes to Divalent Cations To study the diffusion of Mg 2+ and Ca 2+ into liposomes in comparison with the diffusion of Mn 2+, we took advantage of the strict requirement of DNase I by any one
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Fig. 1. Effect of divalent cations and sonication on the activity of pancreatic DNase I. A and B 2 gg of pBR322 DNA and 9 × 10-3 Kunitz units of pancreatic DNase I in 10 mM Tris-HCl, 1 mM NaC1, pH 7, were incubated at 37°C; the enzymatic activity was triggered with a 2 mM solution of (a,b,c) MnC12; (d,e,f) MgC12, and (g,h,i) CaC12. B Pancreatic DNase I was sonicated three times during 5 s in a Lab Supply G112SOI sonicator, as described in Materials and Methods, before the incubation with pBR322 DNA. (j) pBR322 DNA alone sonicated as was indicated for pancreatic DNase I. Numbers are the incubation times in minutes.
of these cations as cofactors. DNA and DNase I were encapsulated in liposomes and the ions were added outside of them. After incubation at 37°C, the extent of DNA hydrolysis inside of liposomes was interpreted as evidence that cations diffused across their lipid membranes. Using this system, unchanged DNA was recovered from liposomes formed only from egg-yolk phosphatidylcholine and incubated at 37°C with 2 mM of either Ca 2÷, Mn 2+, or Mg z÷, for any length of time up to 15 min (Fig. 2a,b,f,j,k), proving that these liposomes were impermeable to divalent cations since DNA was not hydrolyzed. These results also showed that the encapsulation of DNA into liposomes avoided its extensive breakage that sonication produced by itself (Fig. 1B, j). However, when liposomes were formed from eggyolk phosphatidylcholine:egg-yolk phosphatidate (molar ratio 2:1) Ca 2÷ and Mn 2+ really diffused into the vesicles since DNA was digested by DNase I in a way dependent on the incubation time (Fig. 2d,e,h,i). On the contrary, we found that Mg 2+ did not diffuse into liposomes since DNA remained unchanged after 15 min of incubation (Fig. 2m,n). Furthermore, the activity of DNase I was higher inside of liposomes than in the absence of the vesicles (compare d,e and h,i of Fig. 2 with h,i and b,c of Fig. 1). The quantitative evaluation of the DNase I activity inside of vesicles showed that the enzyme was activated 18 times more by Ca 2÷ and only 1.2 times by Mn 2+ in comparison with the ion activation without vesicles (Fig. 3). Figure 2 also shows that liposomes from egg-yolk
563
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0 Fig. 2. Permeabilityof liposomes to divalent cations. Permeability was followed by the hydrolytic activity of DNase I on pBR322 DNA inside the vesicles as described in Materials and Methods. All liposomes were in 10 mM Tris-HC1, 1 mM NaC1, pH 7, and they contained 2 Jag of pBR322 DNA and 9 x l0 3 Kunitz units of pancreatic DNase 1 in their aqueous compartments.Outside the vesicles a 2 mM solution of: (a-e) CaCI2, (f-i) MnCl2, and (j-n) MgC12was added. (a,b,f,j, and k) liposomesfrom egg-yolk phosphatidylcholine(EPC), and (c-e, g-i, and I-n) liposomesfrom egg-yolkphosphatidylcholine:egg-yolkphosphatidate (molar ratio 2: I). Numberswere the incubationtimes in minutes at 37°C.
phosphatidylcholine and from egg-yolk phosphatidylcholine:egg-yolk phosphatidate (molar ratio 2:1) contained practically the same quantity of DNA (compare lanes a and c, f and g, and j and 1), suggesting that both liposomes had the same ability to encapsulate DNA and that they probably had a similar number of lipid bilayers. Lower concentrations of egg-yolk phosphatidate did not permit the activation of DNase I by Ca 2+ and Mn 2+ at 2 mM (not shown). When we used the cations at a concentration of 10 mM, it was also seen that Ca 2+ produced a higher activation of DNase I inside of liposomes than Mn 2+, and that Mg z+ did not activate the enzyme (not shown). Moreover, at 2 mM concentration of cations, the activation of DNase I inside liposomes was lower than at 10 mM (not shown). The stability of liposomes during the permeabilization process was analyzed by adding cations and DNase I outside of the egg-yolk phosphatidylcholine:egg-yolk phosphatidate (2:1 molar ratio) liposomes containing DNA in their aqueous compartments. DNA was not hydrolyzed when Mg 2+ and Ca 2+ were added at 2 mM concentration (Fig. 4A, b,d), in the same way as was described for Mn 2+ (Baeza et al. 1990); furthermore, DNA remained undigested when the cations were used at 10 mM (Fig. 4B, b,c), with the exception of Ca 2+, which triggered a partial hydrolysis of the liposomal DNA (Fig. 4B, d). Total DNA hydrolysis was observed when Triton X-100 was added to these liposome systems (not shown).
I 15
Fig. 3. Quantitativeevaluationof pancreatic DNase I kinetics without vesicles and inside of liposomes. Reactions described in Figs. IA and 2 were evaluated with a gel scanner;DNA percentage was calculated as indicatedunder Materials and Methods. Hydrolyticactivity of pancreatic DNase I without liposomes in the presence of (A) MnCI> (B) MgCI> and (C) CaClz; and of pancreatic DNase I encapsulatedin egg-yolk phosphatidylcholine:egg-yolkphosphatidate (molarratio 2:1) liposomes where (D) MnCI> (E) MgC12, and (F) CaCI2 were added outside the vesicles.
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Fig. 4. Stabilityof liposomes during the permeabilizationprocess. Reactions and concentrationsof DNase I were as indicated in Fig. 2. All liposomes were formed from egg-yolk phosphatidylcholine:eggyolk phosphatidate (molar ratio 2:1) and contained2.5 ~g of pBR322 DNA in their aqueous compartments.DNase I together with divalent cations at 2 mM (A) or 10 mM (B) were added outside of liposomes. (a) Control without cations, (b) MgCI2, (c) MnCI2,and (d) CaCI>
Liposome Leakage Assays The stability of liposomes during the transbilayer movement of cations was also evaluated by analyzing leakage of carboxyfluorescein from the vesicles. Leakage, which leads to dequenching of the dye fluorescence, was monitored in the presence of the three divalent cations. Ves-
564
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Fig. 5. Effectof divalent cation concentrationson the leakage of carboxyfluoresceinfrom liposomes formed from egg-yolkphosphatidylcholine:egg-yolkphosphatidate (molar ratio 2:1). All liposomes contained 70 mM carboxyfluoresceinand divalentcations were added outside of the vesicles. (A) Control without cations, (B) MgC12, (C) MnC12, and (D) CaC12,
icles containing phosphatidate were nonleaky in the absence of cations and when they were added at 2 mM (Fig. 5A-C). Mg 2+ and Mn 2÷ were also nonleaky at 10 mM (Fig. 5B,C); however, at this concentration Ca 2÷ produced a slight leakage effect (Fig. 5D). Liposomes from egg-yolk phosphatidylcholine were nonleaky when the three cations were added at 2 and 10 mM (not shown).
Freeze-Fracture Electron Microscopy Liposomes formed from egg-yolk phosphatidylcholine:egg-yolk phosphatidate (molar ratio 2:1), with and without DNA and DNase I, incubated in the absence of divalent cations at 37°C during 30 rnin and cooled down at 5°C during 1 h before being freeze-fractured, had a size of 100--400 nm and their lipid bilayer presented a smooth surface (Fig. 6A,B), similar to that described for bilayers in the fluid state (Singh et al. 1992). Liposomes incubated in the presence of 10 mM of Mg 2÷ did not modify the size and the structure of their lipid bilayers (Fig. 6C). In contrast, when liposomes were incubated with Ca 2÷, the presence of numerous particles, organized in strings (33 /~ thick) forming a complex network of interconnected ridges on the fracture faces of liposomes (Fig. 6D,E), was observed. The most frequent structures of lipids induced by Mn 2÷ corresponded to strings (37/k thick) organized in pentagonal and hexagonal arrangements (Fig. 6G-J); moreover, the amount of these elements was less compared with those produced by Ca 2÷, in which they seem to cause surface deformations on liposomes (Fig. 6D). Lipidic particles induced by Ca z+ have been previously described in model systems containing dioleoylphosphatidate (Verkleij et al. 1982); however, egg-yolk phosphatidate strings shown in Fig. 6 (D,E) were 33 /~
thick compared with the dioleoylphosphatidate particles that had the average diameter (60-120/~) described by Verkleij (1984); this difference in size could be due to the different fatty acid composition of phosphatidate. It was also shown that practically all the liposomes incubated in the presence of Ca 2+ had lipidic structures (Fig. 6D), whereas approximately 60% of the Mn 2+treated liposomes presented these lipidic transitions and the rest had a smooth surface (Fig. 6H). Moreover, the vesicles incubated with Mg 2÷ and Mn 2÷ did not increase in size compared with the control (Fig. 6A-C,H), whereas the Ca2+-treated liposomes have fused and formed considerably larger vesicles (Fig. 6D). These resuits are in agreement with the fusogenic properties of Ca 2÷ observed on liposomes (Verkeij et al. 1982), and with the behavior of Mg 2÷, which did not induce any fusion of negatively charged vesicles (Portis et al. 1979). When the vesicles were frozen directly from 37°C, without being previously cooled down at 5°C, in the absence of Ca 2+ and Mn 2+, smooth surfaces were observed (not shown); in their presence, they induced different lipidic arrangements. Ca 2÷ seems to modify the regularity of the ridges where particles were apparent (Fig. 6F); with Mn 2+ there were particles (50 A thick) separated 62 A from one another (Fig. 6K). Moreover, at this temperature, Ca 2÷ also induced fusion of liposomes whereas Mn 2+ remained without showing this feature (not shown). When lower concentrations of cations (2 mM) were used, only scarcely lipidic particles induced by Ca 2+ and Mn 2÷ were visualized by freeze-fracture on the liposomes containing egg-yolk phosphatidate which remained without any fusion (not shown). As a control, liposomes from egg-yolk phosphatidylcholine were examined by freeze-fracture under the conditions described for liposomes with phosphatidate, and smooth surfaces were found such as those described in the absence of Ca 2+ and Mn 2+, and in the presence of Mg 2+ (Fig. 6A-C).
31p NMR Spectra Since the interaction of paramagnetic metal ions (Mn 2+, Eu 3÷) with phospholipids completely broadens their 3tp resonance spectrum (Jones et al. 1992), we analyzed the effect of Mn 2÷ in the 3tp NMR spectra of the liposomes from egg-yolk phosphatidylcholine and from egg-yolk phosphatidylcholine:egg-yolk phosphatidate in order to test the Mn 2+ transport mediated by phosphatidate studied in this paper. Furthermore, we obtained the 3tp NMR spectra from both liposomes with and without Mg 2÷ and C a 2+.
Liposomes from egg-yolk phosphatidylcholine:eggyolk phosphatidate, which showed a smooth surface and a size between 100 and 400 nm (see Fig. 6A and 6B), gave a 3tp NMR spectrum (Fig. 7A) with a line shape
565
Fig. 6. Freeze-fractureelectron micrographs of liposomes. Vesicles were prepared by the reverse-phase evaporation method as described in Materials and Methods from egg-yolk phosphatidylcholine:egg-yolkphosphatidate (molar ratio 2:1) in 10 mM Tris-HC1, 1 mM NaCI, pH 7. These vesicles were incubated at 37°C during 30 rain in the absence (A and B) or in the presence of divalent cations 10 mM (C-K): (C) MgCI2, (D-F) CaC12, and (G-K) MnC12. All liposomes were cooled down at 5°C during 1 h before freeze-fracture except for F and K, which were freeze-quenched directly from 37°C. A, D, and H are a low-magnificationoverview of liposomes, at 40,000×; B, E, and I are the corresponding high magnifications at 160,000×. F, G, J, and K are other high magnifications, also at 160,000×, not included in D and It. A r r o w s show egg-yolk phosphatidate particles and strings into the lipid bilayer of liposomes. The shadow direction in all micrographs is from right to left side of the page as indicated by the open arrow in A . Solid lines represent 150 nm in all panels. between the isotropic signal corresponding to small unilamellar vesicles (SUV), with a diameter less than 50 nm, and the anisotropic signal of multilamellar vesicles (MLV), with a diameter greater than 800 nm; moreover, liposomes from egg-yolk phosphatidylcholine also gave a 31p N M R spectrum similar to the vesicles containing egg-yolk phosphatidate (Fig. 7E). After Mn 2÷ at 2 mM concentration was added to the extravesicular solution, the 31p N M R spectrum arising from the egg-yolk phosphatidylcholine:egg-yolk phosphatidate vesicles was broadened beyond detection (Fig. 7B), which suggested the movement of Mn 2÷ through the vesicular membranes; at concentrations of 0.5 mM of Mn 2+, the same effect was observed (Fig. 7B). When 0.5 and 1 mM of Mn 2÷ ions were added to the extravesicular solution of egg-yolk phosphatidylcholine liposomes, there was not a significant change in the 31p signal; however, at 2 mM concentration of Mn 2+, the total signal intensity of the 3tp N M R spectrum was clearly decreased, but without being broadened beyond detection (Fig. 7F); this decrease was more pronounced at 10 mM Mn 2÷ (Fig. 7G). These narrowed signals remained without change over a period of 12 h, indicating that the egg-yolk phosphatidylcholine vesicles were not permeable to Mn 2÷ for at least this period of time.
After 2 mM of Ca 2+ was added to the liposomes containing egg-yolk phosphatidate their 31p N M R signal became sharper (Fig. 7C) than the spectrum without cations (Fig. 7A) and had a slight shift to a low field, indicating that some lipids undergo isotropic motion in the presence of Ca 2+. At 10 mM of Ca 2+, the isotropic 31p N M R was also observed and an increase in the bilayer signal was found (Fig. 7D). No changes in the 31p N M R spectra occurred when liposomes containing egg-yolk phosphatidate were incubated in the presence of 2 and 10 mM of Mg2*-----or when egg-yolk phosphatidylcholine liposomes were added with the same concentrations of Mg 2÷ or Ca 2÷ (not shown).
Discussion In this study, we have demonstrated the transbilayer diffusion of divalent cations into liposomes through the bilayer/nonbilayer phase transitions of phosphatidate, and we simultaneously detected these phase transitions by freeze-fracture and 31p NMR. Liposomes formed from egg-yolk phosphatidylcholine:egg-yolk phosphatidate (molar ratio 2:1) containing pBR322 D N A and DNase I were induced to form, with divalent cations,
566
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CHEMICAL SHIFT (ppm)
Fig. 7. Effectof divalent cations in the 31p NMR spectra of liposomes at 37°C. Samplesof 100/.tmoles of the vesicleswithdrawn from the samplesanalyzedby freeze-fracturewere incubated as described in Materials and Methods. Vesicles from egg-yolk phosphatidylcholine:egg-yolkphosphatidate (molar ratio 2:1) without divalent cations (A) and with 0.5-10 mM MnC12(B) or 2 rnM (C) and 10 mM CaC12 (D). Vesicles from egg-yolkphosphatidylcholinewithout divalent cations (E), and with 2 mM (F) or 10 mM MnC12(G). lipid transitions of phosphatidate which allowed cation diffusion into liposomes; then, this diffusion was measured by the activation of the hydrolysis of DNase I on DNA. Our results clearly showed that the encapsulation of DNA and DNase I inside of the lipid membranes of liposomes avoided the breakage of DNA and the inactivation of the enzyme that ultravibration produced during the formation of the vesicles. Under our experimental conditions, liposomes were permeable to Mn 2+ and Ca 2÷ only when they contained phosphatidate at a 2:1 molar ratio. Otherwise, permeability to cations was absent when liposomes were formed by a lower cone-shaped lipid concentration or using the bilayer preferring lipid egg-yolk phosphatidylcholine alone. Since Mg 2+ is an effective cofactor for free DNase I, the absence of activity of the enzyme encapsulated in the liposomes containing egg-yolk phosphatidate when Mg 2+ ions were added outside of them indicates that liposomes remained intact during their incubation with cations; consequently, the observed permeability to Mn 2÷ and to low concentrations of Ca 2+ (2 raM) cannot be attributed to disruption of liposomes, nor to possible changes in fluidity of the lipid bilayer induced by phosphatidate. Stability of vesicles containing phosphatidate during the transbilayer movement of Mn 2+ and low concentrations of Ca 2÷ (2 raM) was also shown because the vesicles were nonleaky to carboxyfluorescein and because DNase I added outside of preformed liposomes containing DNA could digest this liposomal DNA only when the
vesicles were disrupted with Triton X-100; in contrast, when DNA and DNase I were added outside of empty liposomes, DNA hydrolysis was triggered by the three divalent cations. These experiments also demonstrated that DNA was really encapsulated into liposomes without remaining adsorbed outside of them. However, at 10 mM of Ca 2÷, some liposome disruption was produced during their fusion and DNA was partially exposed to extraliposomal DNase I; liposome disruption by Ca 2÷ was confirmed by carboxyfluorescein leakage from the vesicles. The activity of DNase I was more efficient inside of liposomes containing egg-yolk phosphatidate than in a free solution, probably due to a higher concentration of cations inside the vesicles, as well as a possible reduction in the entropy of the system. The highest activity induced by 2 mM of Ca 2÷ suggested that this ion probably diffused into liposomes faster than Mn 2÷. Freeze-fracture examination of the liposomes permeable to Ca 2+ and Mn 2+ showed bilayer/nonbilayer transitions of phosphatidate and demonstrated a direct correlation between the permeability to cations and the presence of these lipid structures. Since liposomes from egg-yolk phosphatidylcholine alone showed neither lipid structures nor permeability to divalent cations, we can conclude that these structures are phosphatidate transitions necessary to carry out the transbilayer diffusion of Ca 2÷ and Mn 2÷. However, this situation can only be applied to the transport of Ca 2+ at lower concentrations (2 mM), since at higher concentrations (10 raM) this cation produces fusion of liposomes and can also penetrate by membrane disruption. Phosphatidate structures induced by Ca 2÷ and Mn 2÷ were really different in size, number, and arrangement on the surface of liposomes; furthermore, they were also different in function, since those induced by Mn 2÷ did not produce any fusion of liposomes, even at 10 mM concentration; apparently these structures, described for the first time in this work, only participated in the transbilayer transport of Mn 2÷. In contrast, phosphatidate transitions induced by lower concentrations of Ca 2÷ (2 mM) contributed to the transbilayer transport of these ions, whereas those induced by higher concentrations (10 mM) also produced fusion of vesicles. Furthermore, Mg 2÷ ions did not induce any phosphatidate transitions, in agreement with their lack of permeability. The interaction of paramagnetic metal ions, such as Mn 2+, with the phosphate of phospholipids causes pronounced perturbations on the nuclear spin relaxation times which completely broaden their 31p resonance (Sharp 1992). In vesicles, this effect is only obtained when paramagnetic ions interact with the outer and inner lamellae of phospholipids. Then the partial decreased effect of Mn 2÷ in the 31p signal intensity of egg-yolk phosphatidylcholine liposomes together with its total broadened effect in the 31p NMR spectrum of the lipo-
567 somes containing egg-yolk phosphatidate strongly supports the transbilayer transport of Mn 2÷ mediated by phosphatidate. The isotropic motion of lipids detected by the 31p NMR signal of liposomes incubated with Ca 2÷ at 2 mM also demonstrated the presence of phosphatidate transitions, which have a direct correlation with the transbilayer transport of low concentrations of this ion into liposomes; however, the increase in the bilayer signal at 10 mM of Ca 2÷ revealed larger vesicles produced by the fusion due to the higher amount of phosphatidate transitions induced by higher concentrations of this ion. 31p NMR did not show any modification in the dynamics of the phosphate of liposomes incubated with Mg 2+ according to the absence of phosphatidate structures and lack of transport of this cation. Since we have demonstrated in this work that Mn ~÷ is not a fusogenic cation, we propose the use of 3tp NMR spectra as a simple procedure to show the transbilayer transport of Mn 2+ mediated by different cone-shaped lipids. A relevant aspect of our findings was the presence, on the lipid bilayers of liposomes, of phosphatidate structures induced and formed by manganese and calcium that under electron microscopy looked like small protuberances on the surface of the lipid vesicles. These structures have different morphology and functions depending on the cation that induced their formation: phosphatidate transitions formed by manganese participate only in the transbilayer diffusion of this ion, whereas those formed by calcium participate in both its transbilayer diffusion at low cation concentrations and in the fusion of liposomes at higher concentrations; furthermore, the demonstration that magnesium was ineffective in inducing any phosphatidate structures and consequently unable to diffuse into the liposomes and to induce any fusion of the lipid vesicles really validates and confirms the above statements--phosphatidate transitions are necessary for cation diffusion and liposome fusion. We believe that not only transbilayer diffusion of ions and polar substances is important in precellular studies; it is probable that fusion of vesicles could also be an important event in precellular conditions, because fusion processes could have a role in the enrichment of the contents of primitive liposomes. Transbilayer diffusion of cations into closed nonleaky liposomes containing cone-shaped lipids, together with their fusogenic capability, supports the concept that these liposomes could be extraordinarily dynamic vesicles capable of encapsulating and modulating the activity of enzymes in their aqueous compartments in the early Precambrian Earth.
Conclusions
It was demonstrated by freeze-fracture and by 3 t p NMR that liposomes constituted by egg-yolk phosphatidylcholine and the cone-shaped lipid phosphati-
date at a molar ratio of 2:1 formed specific surface phosphatidate arrangements that could be induced by the presence of manganese and calcium ions. . The phosphatidate structures formed by Mn 2+ participated only in the transbilayer diffusion of this cation. In contrast, Ca z+ at low concentrations--in addition to its activity in ionic transport--produced different structural morphologies; at higher concentration (10 raM) it caused fusion of liposomes. Mg 2+ did not form any phosphatidate structures; neither did it participate in ion diffusion and fusion of liposomes. . The liposomes that interchange specific divalent cations with the surrounding environment and have fusogenic properties are dynamic vesicles due to the presence of cone-shaped lipids. These structural and functional properties of liposomes improve in several respects our previous experimental model proposed for the analysis of precellular systems. Acknowledgments.
We thank Dr. J. Or6 for critical reading of the manuscript and for his helpful suggestions and Professor Kenneth E. Davis Gaines for correction of the manuscript. This investigation has been partially supported by DEPI-IPN grant 920673 and by the MacArthur Foundation. C.W. and I.B. were partially supported by DEDICT-COFAA of the IPN.
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