Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca²⁺ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca²⁺-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca²⁺ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca²⁺ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca²⁺ concentration.     

The fusogenic effect of synthetic polycations on negatively charged lipid bilayers

The effect of synthetic polycations, polyallylamine, and polyethylenimine, on liposomes containing phosphatidylserine was investigated along with that of polylysine and divalent cations. The addition of polycations caused aggregation of sonicated vesicles composed of phosphatidylserine and phosphatidylcholine (molar ratio 1:4) as determined by measuring the turbidity changes. Liposomal turbidity increased 10 times compared with that of control liposomes at charge ratios of polymer/vesicle from 0.23 (polylysine) to 2.5 (linear polyethylenimine), while the turbidity was unchanged by the addition of Ca2+ or Mg2+ at charge ratios up to 500. These polycations also induced intermixing of liposomal membranes as indicated by resonance energy transfer between fluorescent lipids incorporated in lipid bilayers, without inducing drastic permeability changes as determined from the calcein release. Fifty percent intermixing of liposomes (0.05 mM as lipid concentration) was induced by these polycations at charge ratios of around 1.0. However, the highest resonance energy transfer was produced by the addition of polyallylamine, which caused multicycles of membrane intermixing between vesicles. Polycation-induced membrane intermixing and permeability changes of phosphatidylserine liposomes were also investigated. At charge ratios of around 1.0, these polymers caused resonance energy transfer of fluorescent lipids incorporated in separate vesicles; however, polyallylamine and branched polyethylenimine also caused permeability increases of liposomal membranes. Membrane intermixing and permeability changes of phosphatidylserine vesicles induced by polyallylamine were dependent on the polymer/vesicle charge ratio, and were different from those induced by Ca2+ since the latter caused half-maximal membrane intermixing or permeability change of phosphatidylserine vesicles at about 1 mM at the liposomal concentrations investigated.     

Proton-induced fusion of oleic acid-phosphatidylethanolamine liposomes

Liposomes composed of oleic acid and phosphatidylethanolamine (3:7 mole ratio) aggregate, become destabilized, and fuse below pH 6.5 in 150 mM NaCl. Fusion is monitored by (i) the intermixing of internal aqueous contents of liposomes, utilizing the quenching of aminonaphthalene-3,6,8-trisulfonic acid (ANTS) by N,N'-p-xylylenebis(pyridinium bromide) (DPX) encapsulated in two separate populations of vesicles, (ii) a resonance energy transfer assay for the dilution of fluorescent phospholipids from labeled to unlabeled liposomes, (iii) irreversible changes in turbidity, and (iv) quick-freezing freeze-fracture electron microscopy. Destabilization is followed by the fluorescence increase caused by the leakage of coencapsulated ANTS/DPX or of calcein. Ca2+ and Mg2+ also induce fusion of these vesicles at 3 and 4 mM, respectively. The threshold for fusion is at a higher pH in the presence of low (subfusogenic) concentrations of these divalent cations. Vesicles composed of phosphatidylserine/phosphatidylethanolamine or of oleic acid/phosphatidylcholine (3:7 mole ratio) do not aggregate, destabilize, or fuse in the pH range 7-4, indicating that phosphatidylserine and phosphatidylcholine cannot be substituted for oleic acid and phosphatidylethanolamine, respectively, for proton-induced membrane fusion. Freeze-fracture replicas of oleic acid/phosphatidylethanolamine liposomes frozen within 1 s of stimulation with pH 5.3 display larger vesicles and vesicles undergoing fusion, with membrane ridges and areas of bilayer continuity between them. The construction of pH-sensitive liposomes is useful as a model for studying the molecular requirements for proton-induced membrane fusion in biological systems and for the cytoplasmic delivery of macromolecules.     

Fusion of liposomes containing a novel cationic lipid, N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles

The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) and either phosphatidylcholine (PC) or phosphatidylethanolamine (PE) has been investigated by a fluorescence resonance energy transfer assay for lipid mixing, dynamic light scattering, and electron microscopy. Polyvalent anions induced the fusion of DOTMA/PE (1:1) liposomes with the following sequence of effectiveness: citrate greater than EDTA greater than phosphate, in the presence 100 mM NaCl, pH 7.4. Sulfate, dipicolinate, and acetate were ineffective. DOTMA/PC (1:1) vesicles were completely refractory to fusion in the presence of multivalent anions in the concentration range studied, consistent with the inhibitory effect of PC in divalent cation induced fusion of negatively charged vesicles. DOTMA/PE vesicles could fuse with DOTMA/PC vesicles in the presence of high concentrations of citrate, but not of phosphate. Mixing of DOTMA/PE liposomes with negatively charged phosphatidylserine (PS)/PE or PS/PC (1:1) vesicles resulted in membrane fusion in the absence of multivalent anions. DOTMA/PC liposomes also fused with PS/PE liposomes and, to a limited extent, with PS/PC liposomes. These observations suggest that the interaction of the negatively charged PS polar group with the positively charged trimethylammonium of DOTMA is sufficient to mediate fusion between the two membranes containing these lipids and that the nature of the zwitterionic phospholipid component of these vesicles is an additional determinant of membrane fusion.     

Polyphosphoinositide inclusion in artificial lipid bilayer vesicles promotes divalent cation-dependent membrane fusion.

Recent studies suggest that phosphoinositide kinases may participate in intracellular trafficking or exocytotic events. Because both of these events ultimately require fusion of biological membranes, the susceptibility of membranes containing polyphosphoinositides (PPIs) to divalent cation-induced fusion was investigated. Results of these investigations indicated that artificial liposomes containing PPI or phosphatidic acid required lower Ca2+ concentrations for induction of membrane fusion than similar vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylcholine. This trend was first observed in liposomes composed solely of one type of phospholipid. In addition, however, liposomes designed to mimic the phospholipid composition of the endofacial leaflet of plasma membranes (i.e., liposomes composed of combinations of PPI, phosphatidylethanolamine, and phosphatidylcholine) also required lower Ca2+ concentrations for induction of aggregation and fusion. Liposomes containing PPI and phosphatidic acid also had increased sensitivity to Mg(2+)-induced fusion, an observation that is particularly intriguing given the intracellular concentration of Mg2+ ions. Moreover, the fusogenic effects of Ca2+ and Mg2+ were additive in vesicles containing phosphatidylinositol bisphosphate. These data suggest that enzymatic modification of the PPI content of intracellular membranes could be an important mechanism of fusion regulation.     

Annexin-mediated membrane fusion of human neutrophil plasma membranes and phospholipid vesicles.

Membrane fusion was studied using human neutrophil plasma membrane preparations and phospholipid vesicles approximately 0.15 microns in diameter and composed of phosphatidylserine and phosphatidylethanolamine in a ratio of 1 to 3. Liposomes were labeled with N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) and lissamine rhodamine B derivatives of phospholipids. Apparent fusion was detected as an increase in fluorescence of the resonance energy transfer donor, NBD, after dilution of the probes into unlabeled membranes. 0.5 mM Ca2+ alone was sufficient to cause substantial fusion of liposomes with a plasma membrane preparation but not with other liposomes. Both annexin I and des(1-9)annexin I caused a substantial increase in the rate of fusion under these conditions while annexin V inhibited fusion. Fusion mediated by des(1-9)annexin I was observed at Ca2+ concentrations as low as approximately 5 microM, suggesting that the truncated form of this protein may be active at physiologically low Ca2+ concentrations. Trypsin treated plasma membranes were incapable of fusion with liposomes, suggesting that plasma membrane proteins may mediate fusion. Liposomes did not fuse with whole cells at any Ca2+ concentration, indicating that the cytoplasmic side of the membrane is involved. These results suggest that annexin I and unidentified plasma membrane proteins may play a role in Ca(2+)-dependent degranulation of human neutrophils.     

Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles

We have investigated the contribution of various phospholipids to membrane fusion induced by divalent cations. Fusion was followed by means of a new fluorescence assay monitoring the mixing of internal aqueous contents of large (0.1 μm diameter) unilamellar liposomes. The rate and extent of fusion induced by Ca²⁺ in mixed phosphatidylserine/phosphatidylcholine vesicles were lower compared to those in pure phosphatidylserine vesicles. The presence of 50% phosphatidylcholine completely inhibited fusion, although the vesicles aggregated upon Ca²⁺ addition. When phosphatidylserine was mixed with phosphatidylethanolamine, however, rapid fusion could be induced by Ca²⁺ even in mixtures that contained only 25% phosphatidylserine. Phosphatidylethanolamine also facilitated fusion by Mg²⁺ which could not fuse pure phosphatidylserine vesicles. In phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine mixtures, in which the phosphatidylcholine content was kept at 25%, phosphatidylethanolamine could not substitute for phosphatidylserine, and the fusogenic capacity of Mg²⁺ was abolished by the presence of merely 10% phosphatidylcholine. The initial rate of release of vesicle contents was slower than the rate of fusion in all the mixtures used. The presence of phosphate effected a considerable decrease in the threshold concentration of Ca²⁺ and also enhanced     

Interaction of a peripheral protein of the erythrocyte membrane, band 4.1, with phosphatidylserine-containing liposomes and erythrocyte inside-out vesicles

Interactions of band 4.1 with mixed phospholipid membranes [phosphatidylserine (PtdSer), phosphatidylethanolamine, phosphatidylcholine, etc.] and erythrocyte inside-out vesicles were studied. Band 4.1 showed a higher affinity to PtdSer-containing membranes. The amount of binding to PtdSer-containing liposomes was larger than that to PtdSer-lacking liposomes. The amount of binding to inside-out vesicles did not change significantly on a protease treatment of the vesicles. The amount of band 4.1 bound on inside-out vesicles decreased on PtdSer-decarboxylase treatment of the vesicles. Ca2+ acted inhibitory to the binding of band 4.1. Band 4.1 together with PtdSer-containing vesicles but not with PtdSer-lacking vesicles induced gelation of spectrin-actin copolymer solution. Ca2+ inhibited the gelation. Fluorescence energy transfer from PtdSer-containing vesicles to band 4.1 was larger than that from PtdSer-lacking vesicles. Band 4.1 caused a marked release of tempocholine from preloaded PtdSer-containing liposomes but not from PtdSer-lacking liposomes. The release was larger from liposomes containing more PtdSer. Ca2+ was inhibitory to the tempocholine release. We suggest from these results that band 4.1 provides another anchoring site for the cytoskeletal spectrin-actin network to PtdSer domains in the inner layer of erythrocyte membrane. This anchoring may be involved in functional regulation since the interaction causes the membrane permeability change that is dependent on Ca2+.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monitoring of phospholipid vesicle fusion by fluorescence energy transfer between membrane-bound dye labels

A sensitive method which utilizes fluorescence energy transfer to assay Ca2+ -or Mg2+ -mediated fusion of phospholipid vesicles is reported. More than 85% quenching results when phosphatidylserine vesicles labelled with dansyl phosphatidylethanolamine (donor) are fused with vesicles labelled with rhodamine phosphatidylethanolamine (acceptor) in the presence of 5 mM CaCl2 or 10 mM MgCl2. Higher concentrations of divalent cations are required to obtain maximal quenching when phosphatidylserine is partially replaced with phosphatidylethanolamine or phosphatidylcholine. The rate of vesicle fusion is dependent upon the concentrations of both cation and vesicles. Maximum quenching occurs within 5 min using phosphatidylserine vesicles and 5 mM Ca2+, but quenching is incomplete even after 20 h with 0.8--2 mM Ca2+. This probably reflects the heterogeneous size distribution of these vesicles, since the extent of fusion was found to correlated with vesicle size. Binding of antibody to membrane-localized phenobarbital hapten effectively blocks Ca2+ -mediated vesicle fusion. This effect can be inhibited by preincubation of the antibody with phenobarbital. Leakage of tempocholine from intact vesicles induced by 5 mM Ca2+ occurs even when fusion is prevented by bound antibody. This demonstrates that fusion is not a necessary requirement for Ca2+ -induced leakage.     

Fusion of phosphatidylserine and mixed phosphatidylserine-phosphatidylcholine vesicles. Dependence on calcium concentration and temperature

Dynamic light scattering has been used to study the temperature dependence of Ca2+-induced fusion of phosphatidylserine vesicles and mixed vesicles containing phosphatidylserine and different phosphatidylcholines. The final vesicle size after Ca2+ and EDTA incubation serves as a measure of the extent of fusion. With phosphatidylserine vesicles, the extent of fusion shows a sharp maximum at an incubation temperature which depends on the Ca2+ concentration between 0.8 and 2 mM. The shift in the fusion peak temperature with Ca2+ concentration is similar to the typical shift in the phase transition temperature with divalent cation concentration in acidic phospholipids. The results suggest a direct correlation between the fusion peak temperature and the phase transition temperature in the presence of Ca2+ prior to fusion. With mixed vesicles containing up to 33% of a phosphatidylcholine in at least 2 mM Ca2+, the extent of fusion as a function of incubation temperature also shows a maximum. The fusion peak temperature is essentially independent of the quantity and type of phosphatidylcholine and the Ca2+ concentration, and identical to that with pure phosphatidylserine in excess Ca2+. The results imply that Ca2+- induced molecular segregation occurs first, and fusion subsequently takes place between pure phosphatidylserine domains.     

Polyamines as modulators of membrane fusion: aggregation and fusion of liposomes

We have studied the effect of the polyamines (spermine, spermidine, and putrescine) on the aggregation and fusion of large (approximately 100 nm in diameter) unilamellar liposomes in the presence of 100 mM NaCl, pH 7.4. Liposome fusion was monitored by the Tb/dipicolinic acid fluorescence assay for the intermixing of internal aqueous contents, and the release of contents was followed by carboxyfluorescein fluorescence. Spermine and spermidine at physiological concentrations aggregated liposomes composed of pure phosphatidylserine (PS) or phosphatidate (PA) and mixtures of PA with phosphatidylcholine (PC) but did not induce any fusion. However, liposomes composed of mixtures of acidic phospholipids, cholesterol, and a high mole fraction of phosphatidylethanolamine could be induced to fuse by spermine and spermidine in the absence of divalent cations. Putrescine alone in the physiological concentration range was ineffective for both aggregation and fusion of these liposomes. Liposomes made of pure PC did not aggregate in the presence of polyamines. Addition of aggregating concentrations of spermine caused a drastic increase in the rate of Ca(2+)-induced fusion of PA liposomes and a large decrease in the threshold Ca(2+) concentration required for fusion. This effect was less pronounced in the case of PS or PA/PC vesicles. Preincubation of PA vesicles with spermine before the addition of Ca(2+) resulted in a 30-fold increase in the initial rate of fusion. We propose that polyamines may be involved in the regulation of membrane fusion phenomena accompanying cell growth, cell division, exocytosis, and fertilization.     

Leakage of internal markers from erythrocytes and lipid vesicles induced by melittin, gramicidin S and alamethicin: a comparative study.

The membrane-disruptive capacities of melittin, derivatised melittins, alamethicin and gramicidin S have been compared for the human erythrocyte membrane and lipid vesicles of three different compositions (phosphatidylcholine, 85% phosphatidylcholine/15% phosphatidylserine, and a lipid analogue of the outer leaflet of the human erythrocyte membrane). The sensitivity to ionic strength, divalent metal ions and polylysine of release of fluorescent markers from liposomes and of haemoglobin from intact erythrocytes has been assayed. Acetyl melittin was found to he more effective than melittin in lysing phosphatidylcholine and phosphatidylcholine/phosphatidylserine vesicles, somewhat less effective in the lipid analogue and markedly less effective in lysing erythrocytes. Succinyl melittin was non-haemolytic, but was able to lyse lipid vesicles at a high concentration. Ca2+ inhibited melittin haemolysis at high ionic strength (150 mM NaCl), but produced a more complex response of stimulation followed by inhibition at low ionic strength. In lipid vesicles, Ca2+ either stimulated melittin lysis or was ineffective. Zn2+ exerted effects similar to Ca2+ with lipid vesicles at approx. 10-fold lower concentration except that a weak inhibition was observed for the erythrocyte membrane lipid analogue at high ionic strength. Polylysine strongly inhibited haemolysis by melittin at low ionic strength, but was ineffective or stimulatory in lipid vesicle lysis. High phosphate concentration also inhibited melittin haemolysis, but again no corresponding effect could he found in any of the lipid vesicle systems. These disparities between effects of melittin on erythrocytes and lipid vesicles support the proposal that melittin-protein interactions are of consequence to its haemolytic action. Similar experiments were performed with gramicidin S and alamethicin in order to compare their lytic properties with those of melittin. It was found that each lysin exhibited its own individual pattern of sensitivity to lipid composition, ionic strength and inhibition by cations. It thus appears likely that the detailed molecular interactions responsible for lysis are significantly different for each of these three agents.     

Similarity in calcium channel activity of annexin V and matrix vesicles in planar lipid bilayers.

Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation in growing bone, are rich in annexin V. When MVs are fused with planar phospholipid bilayers, a multiconductance Ca2+ channel is formed, with activity essentially identical to that observed when annexin V is delivered to the bilayer with phosphatidylserine liposomes. Ca2+ currents through this channel, from either MV or annexin V liposomes, are blocked by Zn2+, as is Ca2+ uptake by MV incubated in synthetic cartilage lymph. Blockage by Zn2+ was most effective when applied to the side containing the MV or liposomes. ATP and GTP differentially modulated the activity of this channel: ATP increased the amplitude of the current and the number of conductance states; GTP dramatically reduced the number of events and conductance states, leading to well-defined Ca2+ channel activity from either MV or the annexin V liposomes. In the distinctive effects of ATP, GTP, and Zn2+ on the Ca2+ channel activity observed in both the MV and the liposome systems, the common factor was the presence of annexin V. From this we conclude that Ca2+ entry into MV results from the presence of annexin V in these membrane-enclosed structures.     

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.     

Reconstitution of rabbit thrombomodulin into phospholipid vesicles

The influence of phospholipid on thrombin-thrombomodulin-catalyzed activation of protein C has been studied by incorporating thrombomodulin into vesicles by dialysis from octyl glucoside-phospholipid mixtures. Thrombomodulin was incorporated into vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (30% phosphatidylserine and 70% phosphatidylcholine). Thrombomodulin is randomly oriented in vesicles of different phospholipid composition. Incorporation of thrombomodulin into phosphatidylcholine, with or without phosphatidylserine, alters the Ca2+ concentration dependence of protein C activation. Soluble thrombomodulin showed a half-maximal rate of activation at 580 microM Ca2+, whereas half-maximal rates of activation of liposome-reconstituted thrombomodulin were obtained between 500 microM Ca2+ and 2 mM Ca2+, depending on the composition (protein:phospholipid) of the liposomes. The Ca2+ dependence of protein C activation fits a simple hyperbola for the soluble activator, while the Ca2+ dependence of the membrane-associated complex is distinctly sigmoidal with a Hill coefficient greater than 2.4. In contrast, the Ca2+ dependence of gamma-carboxyglutamic acid (Gla) domainless protein C activation is unchanged by membrane reconstitution (1/2 max = 53 +/- 10 microM) and fits a simple rectangular hyperbola. Incorporation of thrombomodulin into pure phosphatidylcholine vesicles reduces the Km for protein C from 7.6 +/- 2 to 0.7 +/- 0.2 microM. Increasing phosphatidylserine to 20% decreased the Km for protein C further to 0.1 +/- 0.02 microM. Membrane incorporation has no influence on the activation of protein C from which the Gla residues are removed proteolytically (Km = 6.4 +/- 0.5 microM). The Km for protein C observed on endothelial cells is more similar to the Km observed when thrombomodulin (TM) is incorporated into pure phosphatidylcholine vesicles than into negatively charged vesicles, suggesting that the protein C-binding site on endothelial cells does not involve negatively charged phospholipids. In support of this concept, we observed that prothrombin and fragment 1, which bind to negatively charged phospholipids, do not inhibit protein C activation on endothelial cells or TM incorporated into phosphatidylcholine vesicles, but do inhibit when TM is incorporated into phosphatidylcholine:phosphatidylserine vesicles. These studies suggest that neutral phospholipids lead to exposure of a site, probably on thrombomodulin, capable of recognizing the Gla domain of protein C.     

Purification and Mode of Action of Synexin: A Protein Enhancing Calcium-Induced Membrane Aggregation

Synexin, a protein from the cytosol of the adrenal medulla, selectively increases the ability of Ca2+ to aggregate chromaffin granules and other membrane-bound particles. The ability of synexin to self-aggregate in the presence of Ca2+ can be employed in the purification of the protein by monitoring purification with parallel assays that utilize the aggregation of both chromaffin granule membranes and phosphatidylserine liposomes. It is shown that the enhancement of the Ca2+-induced aggregation of both liposomes and chromaffin granule membranes is a property associated with a 47,000 Mr protein. Trypsin inactivated synexin. We found that if granule membranes were well washed after trypsin treatment, they were still excellent substrates for synexin aggregation. This finding cannot be explained by extinction changes owing to synexin self-aggregation. The 47,000 Mr protein enhancement Ca2+ aggregation of phosphatidylserine liposomes containing up to 40% phosphatidylcholine, liposomes made from lipids extracted from chromaffin granule membranes, and trypsin-treated chromaffin granule membranes, thus suggesting that synexin activity in vivo may be independent of specific membrane proteins but dependent on the presence of acidic phospholipids in the membrane.     

Fusion of phospholipid vesicles reconstituted with cytochrome c oxidase and mitochondrial hydrophobic protein.

Reconstituted cytochrome oxidase liposomes were fused with liposomes reconstituted with mitochondrial hydrophobic protein, which acts as a membrane-bound uncoupler of cytochrome oxidase. Fusion was assayed by the loss of respiratory control of cytochrome oxidase as measured by the increased rate of ascorbate oxidation induced by hydrophobic protein when both proteins shared the same vesicles. Fusion was dependent on the presence of phosphatidylserine in the liposomes Ca++ in the aqueous medium. Phosphatidylcholine-phosphatidylserine liposomes required higher concentrations of phosphatidylserine and Ca++ than did phosphatidylethanolamine-phosphatidylserine liposomes. Cytochrome oxidase vesicles containing high concentrations of phosphatidylserine showed little or no respiratory control, while those with lower concentrations showed high respiratory control; respiratory control could be induced by fusing cytochrome oxidase vesicles containing high phosphatidylserine with protein-free liposomes containing low phosphatidylserine concentration. If cytochrome oxidase vesicles and hydrophobic protein vesicles were prefused separately for 15 min, they lost the ability to fuse upon being subsequently mixed together. The reconstituted vesicles had diameters of about 200 A; fusion yielded vesicles with diameters in excess of 1000 A.     

Role of synexin in membrane fusion. Enhancement of calcium-dependent fusion of phospholipid vesicles

Synexin, a soluble adrenal medullary and liver protein which causes calcium-dependent aggregation of isolated chromaffin granules, was isolated and purified according to published procedures. The effects of synexin on the kinetics of membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Synexin lowers the threshold of CA2+ concentration required for fusion of large unilamellar vesicles of phosphatidylserine and a mixture of phosphatidylserine with phosphatidylethanolamine. synexin also increases drastically the initial rate of fusion. the initial rate of fusion increases with the quantity of synexin present in the reaction mixture. In the presence of 1-2 mM Ca2+ and 50 microM phospholipid, synexin at 20 to 40 micrograms/ml increases the rate of fusion by two orders of magnitude. Mg2+ does not support synexin-induced fusion. With vesicles containing a mixture of phosphatidylserine with phosphatidylcholine, synexin enhances aggregation in the presence of CA2+, without promoting fusion. Synexin may play a role in exocytosis by promoting fusion of membranes containing specific phospholipids in the presence of Ca2+.     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40 degrees C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca2+, the additional component was removed by an electrostatic interaction between Ca2+ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40 degrees C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.