Evaluation of membrane phase behavior as a tool to detect extrinsic protein-induced domain formation: binding of prothrombin to phosphatidylserine/phosphatidylcholine vesicles

The temperature-composition phase diagram of mixed dimyristoylphosphatidylserine (DMPS) and dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles was determined in the presence and absence of bound bovine prothrombin by monitoring the phospholipid order-disorder phase separation using diphenylhexatriene (DPH) fluorescence anisotropy. The shape of the membrane temperature-composition diagram was essentially unaltered by the binding of prothrombin in the presence of Ca2+ although the two-phase (gel/fluid) region was slightly narrowed and shifted by 1-10 degrees C to higher temperatures. This result does not support the popular idea that extensive domains rich in negatively charged phospholipid are induced in response to prothrombin binding. Instead of implying domain formation, our results demonstrate that the observed increase in melting temperature associated with binding of prothrombin to acidic phospholipid membranes can be accounted for by the observed altered membrane order both in the fluid and in the solid lamellar phases. The membrane order in the liquid-crystalline phase increased with increased acidic lipid content, and much more so for DMPS than for dipentadecanoylphosphatidylglycerol (DC15PG). These results demonstrate that simple shifts in membrane phase behavior cannot be properly interpreted to prove the existence of charged lipid domains. In addition, we report the unexpected observation that prothrombin increased the anisotropy of DPH in DMPS/DMPC vesicles in the liquid-crystalline phase in the absence of Ca2+ as well as in its presence. This effect was seen to a lesser extent and only at a much higher charged-lipid content for DC15PG/DMPC vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of membrane fusion by membrane fluidity: temperature dependence of divalent cation induced fusion of phosphatidylserine vesicles

We have investigated the temperature dependence of the fusion of phospholipid vesicles composed of pure bovine brain phosphatidylserine (PS) induced by Ca2+ or Mg2+. Aggregation of the vesicles was monitored by 90 degrees light-scattering measurements, fusion by the terbium/dipicolinic acid assay for mixing of internal aqueous volumes, and release of vesicle contents by carboxyfluorescein fluorescence. Membrane fluidity was determined by diphenylhexatriene fluorescence polarization measurements. Small unilamellar vesicles (SUV, diameter 250 A) or large unilamellar vesicles (LUV, diameter 1000 A) were used, and the measurements were done in 0.1 M NaCl at pH 7.4. The following results were obtained: (1) At temperatures (0-5 degrees C) below the phase transition temperature (Tc) of the lipid, LUV (PS) show very little fusion in the presence of Ca2+, although vesicle aggregation is rapid and extensive. With increasing temperature, the initial rate of fusion increases dramatically. Leakage of contents at the higher temperatures remains limited initially, but subsequently complete release occurs as a result of collapse of the internal aqueous space of the fusion products. (2) SUV (PS) are still in the fluid state down to 0 degree C, due to the effect of bilayer curvature, and fuse rapidly in the entire temperature range from 0 to 35 degrees C in the presence of Ca2+. The initial rate of leakage is low relative to the rate of fusion. At higher temperatures (15 degrees C and above), subsequent collapse of the vesicles' internal space causes complete release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of dextran sulfate with phospholipid surfaces and liposome aggregation and fusion

The binding of dextran sulfate to phospholipid liposomes was investigated by microelectrophoresis experiments. The polyanion binds to neutral phospholipid liposomes (DMPC and PE) only in the presence of Ca2+. If positively charged stearylamine is incorporated in the vesicles dextran sulfate is bound without Ca2+. Negatively charged phospholipids as PS do not bind dextran sulfate, even in the presence of millimolar concentrations of Ca2+. The adsorption of dextran sulfate results in an aggregation of vesicles due to a bridging mechanism. In all cases the aggregation is followed by a disaggregation toward higher dextran sulfate concentrations. The disaggregation process starts at polymer concentrations smaller than the concentration of the onset of saturation of the adsorption. By use of the probe dilution method a fusion of small DMPC and DMPC/PE vesicles in the presence of Ca2+ and dextran sulfate was found.     

Monovalent cation-induced fusion of acidic phospholipid vesicles

Fusion of small unilamellar vesicles (SUV) consisting of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and phosphatidylglycerol (PG) from egg yolk, dipalmitoylphosphatidylserine (DPPS) and phosphatidylserine (PS) from bovine brain was studied as a function of monovalent cation concentration. Fusion was detected by measuring the changes in the excimer to monomer fluorescence intensity ratio (IE/M) of pyrene-labeled phospholipid analogues upon fusion of the pyrene-labeled and unlabeled vesicles. No fusion was observed from vesicles consisting of DMPC, PS from bovine brain or PG from egg yolk upon addition of NaCl (up to 1 M). However, considerable fusion was evident for vesicles consisting of DMPG or DPPS upon addition of monovalent cations (300 mM to 1 M). Fusion kinetics were fast reaching a plateau after 5 min of addition of cations. The order of efficiency of different monovalent cations to induce the fusion of DMPG vesicles as judged by the changes of the IE/M ratio was Li+ greater than Na+ greater than K+ greater than Cs+. DSC-scan of sonicated DMPG vesicles showed, in the absence of salt, a phase transition at 19.2 degrees C with enthalpy of 1.1 kcal.mol-1. After incubation in the presence of 600 mM NaCl the DSC scan showed a narrow phase transition at 24.1 degrees C with enthalpy of 6.9 kcal.mol-1 and a pronounced pretransition, both supporting that the fusion of the vesicles had occurred in the presence of NaCl. The results indicate that sonicated vesicles consisting of acidic phospholipids with fully saturated fatty acids fuse in the presence of monovalent cations, whereas those containing unsaturated fatty acids do not.     

Membrane binding induces lipid-specific changes in the denaturation profile of bovine prothrombin. A scanning calorimetry study.

Prothrombin denaturation was examined in the presence of Na2EDTA, 5mM CaCl2, and CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-phosphatidylglycerol (DOPG). Heating denaturation of prothrombin produced thermograms showing two peaks, a minor one at approximately 59 degrees C previously reported to correspond to denaturation of the fragment 1 region (Ploplis, V. A., D. K. Strickland, and F. J. Castellino 1981. Biochemistry. 20:15-21), and a main one at approximately 57-58 degrees C, reportedly due to denaturation of the rest of the molecule (prethrombin 1). The main peak was insensitive to the presence of 5mM Ca2+ whereas the minor peak was shifted to higher temperature (Tm approximately 65 degrees C) by Ca2+. Sufficient concentrations of POPC/bovPS (75/25) large unilamellar vesicles to guarantee binding of 95% of prothrombin resulted in an enthalpy loss in the main endotherm and a comparable enthalpy gain in the minor endotherm accompanying an upward shift in peak temperature (Tm approximately 73 degrees C). Peak deconvolution analysis on the prothrombin denaturation profile and comparison with isolated prothrombin fragment 1 denaturation endotherms suggested that the change caused by POPC/PS vesicles reflected a shift of a portion of the enthalpy of the prethrombin 1 domain to higher temperature (Tm approximately 77 degrees C). The enthalpy associated with this high-temperature endotherm increased in proportion to the surface concentration of PS. By contrast, POPC/DOPG (50/50) membranes shifted the prethrombin 1 peak by 4 degrees C to a lower temperature and the fragment 1 peak by 5 degrees C to a higher temperature. The data lead to a hypothesis that the fragment 1 and prethrombin 1 domains of prothrombin do not denature quite independently and that binding of prothrombin to acidic-lipid membranes disrupts the interaction between these domains. It is further hypothesized that PS containing membranes exert the additional specific effect of decoupling the denaturation of two subdomains of the prethrombin 1 domain of prothrombin.     

Comparison of lipid binding and kinetic properties of normal, variant, and gamma-carboxyglutamic acid modified human factor IX and factor IXa

The abilities of normal and three abnormal factor IXa molecules to activate factor X and to bind to phospholipid membranes have been compared to define the contributions of protein-lipid interactions and factor IXa light chain-heavy chain interactions to the functioning of this protein. The abnormal proteins studied had altered amino acid residues in their light chains. The heavy-chain regions, containing the active site serine and histidine residues, were normal in the abnormal proteins on the basis of titration by antithrombin III. The binding constants (Kd) for normal (N), variant [Chapel Hill (CH) and Alabama (AL)], and gamma-carboxyglutamic acid (Gla) modified (MOD) factors IX and IXa to phosphatidylserine (PS)/phosphatidylcholine (PC) small, unilamellar vesicles (SUV) were measured by 90 degrees light scattering. The Kd values for factor IXN binding were quite sensitive to the PS content of the membrane but less sensitive to Ca2+ concentrations between 0.5 and 10 mM. The zymogen and activated forms of both normal and abnormal factor IX bound with similar affinities to PS/PC (30/70) SUV. In the cases of factor IXaN and factor IXaAL, but not factor IXaCH or factor IXaMOD, irreversible changes in scattering intensity suggested protein-induced vesicle fusion. Since the activation peptide is not released from factor IXaCH, the normal interaction of factor IXa with a membrane must require the release of the activation peptide and the presence of intact Gla residues. The rate of factor X activation by normal and abnormal factor IXa was obtained by using a chromogenic substrate for factor Xa in the presence of PS/PC (30/70) SUV and 5 mM Ca2+.     

Spontaneous fusion of phosphatidylcholine small unilamellar vesicles in the fluid phase

Using a high-sensitivity differential scanning microcalorimeter capable of performing cooling scans, we have examined the phase behavior of small unilamellar vesicles (SUV) as a function of time of storage above their order-disorder phase transition. Vesicles composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were examined. Cooling scans on fresh (5-7-h postsonication) samples revealed broad, relatively simple heat capacity peaks (peak temperatures: 19.9 degrees C for DMPC, 37.8 degrees C for DPPC) free of high-temperature spikes or shoulders. Subsequent heating scans displayed a sharp peak characteristic of previously described fusion products formed below the phase transition. SUV samples stored for 1 or more days above their phase transition displayed a moderately broad, high-temperature shoulder (23.8 degrees C for DMPC and 40.2 degrees C for DPPC) in the cooling profile. For DMPC, the enthalpy associated with this peak increased in a first-order fashion with time. Hydrolysis products were not detected until 12-20 days of storage. Both the rate and extent of shoulder appearance increased with temperature (k = 0.0017 h-1, fraction of total enthalpy = 0.1 at 36 degrees C; k = 0.0037 h-1, fraction = 0.2 at 42 degrees C). Freeze-fracture electron micrographs confirmed that an intermediate-sized vesicle population (diameters 400-500 A) appeared in SUV samples stored above their phase transition. Also, the trapped volume of DMPC SUV increased from 0.26 microL/mumol after 17 h of storage to 0.54 microL/mumol after storage for 16 days at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid lateral organization in synthetic membranes as monitored by pyrene-labeled phospholipids: effects of temperature and prothrombin fragment 1 binding

Pyrene-labeled phospholipids have been used to test for the existence of lateral domains due to temperature-induced phase separations and binding of prothrombin fragment 1 to charged lipid vesicles. When in close proximity, pyrene-containing probes can exchange excited-state energy to form excimers; the ratio of the excimer to monomer fluorescence intensity (E/M) is proportional to the local concentration of probe in the membranes, as well as to the excimer lifetime and the probe's lateral diffusion coefficient. The ability of the pyrene-labeled phospholipids to quantitatively report the coexistence of multiple environments was demonstrated in dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylcholine multilamellar vesicle preparations of varying compositions, each of which contained coexisting fluid and gel phases. In this system, pyrene-labeled phosphatidylcholine was found to favor the fluid relative to the gel phase with a partition coefficient of 7. At 37 degrees C, in dioleoylphosphatidylglycerol (DOPG)/palmitoyloleoylphosphatidylcholine (POPC) large, unilamellar vesicles containing either pyrene-labeled phosphatidylglycerol (py-PG) or pyrene-labeled phosphatidylcholine (py-PC), the excimer lifetime (37 ns) and the lateral diffusion constant of the probe (5.8 X 10(-8) cm2/s) were independent of the membrane composition and the presence of fragment 1 and Ca2+. Consequently, E/M was directly proportional to only the local concentration of the py-PG or py-PC probes. When saturating amounts of fragment 1 and 5 mM Ca2+ were added to DOPG/POPC vesicles that contained either probe, no change in E/M and hence the local probe concentration was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of sphingomyelins and phosphatidylcholines with fluorescent dehydroergosterol

The fluorescent sterol dehydroergosterol was used as a cholesterol analogue in conjunction with multifrequency phase and modulation (1-250 MHz) fluorometry to examine whether sterols (1) interact preferentially with fluid- or solid-phase phospholipids and (2) interact preferentially with sphingomyelin in phase-separated or phase-miscible cosonicated phospholipid membranes. Cosonicated small unilamellar vesicles (SUV) were produced by mixing lipids in organic solvents, drying the mixture, adding buffer, sonicating, and separating SUV. Phospholipids of synthetic as well as biological origin were utilized. In phase-separated, cosonicated SUV of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC, 1:1 molar ratio), the fluorescent sterol (0.5 mol %) interacted preferentially with the fluid-phase lipid (partition coefficient, Kf/s = 2.6-3.4) according to four criteria. First, dehydroergosterol detected only the phase transition of DMPC, the phospholipid with the lower phase transition temperature. Second, the dehydroergosterol fluorescence polarization, limiting anisotropy, order parameter, and rotational relaxation time in the cosonicated vesicle were similar to those of dehydroergosterol in SUV composed only of DMPC. Third, the number of dehydroergosterol fluorescence lifetime components as well as the distribution in the cosonicated SUV was similar to that of dehydroergosterol in SUV composed of DMPC. Fourth, dehydroergosterol concentration-dependent self-quenching was detected in DSPC SUV at much lower dehydroergosterol concentration than in DMPC SUV. Preference of dehydroergosterol for fluid-phase lipids was also observed by monitoring dehydroergosterol exchange between individually sonicated DMPC SUV and DSPC SUV after the two types of vesicles were mixed in equal proportions. In these SUV mixtures, the dehydroergosterol also partitioned into the more fluid SUV, 99:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that the thrombin-catalyzed feedback cleavage of fragment 1.2 at Arg154-Ser155 promotes the release of thrombin from the catalytic surface during the activation of bovine prothrombin

During the course of prothrombin activation, as catalyzed by Factor Xa, Factor Va, Ca2+, and negatively-charged phospholipid vesicles, the three proteins distribute between the fluid phase and the vesicle surface. On the vesicle, efficient Factor Xa-catalyzed proteolysis yields thrombin plus Fragment 1.2. Further thrombin-catalyzed feedback cleavage of the latter then yields Fragment 1 plus Fragment 2. Prior to this cleavage Fragment 1.2 might retain thrombin at the site of catalysis since it binds both phospholipid and thrombin through its respective Fragment 1 and Fragment 2 domains. In order to study the role of the feedback cleavage, light scattering at right angles was used to deduce the nature of the components associated with the vesicle during prothrombin activation by continuous monitoring of the relative molecular weight of the vesicle-protein complex. When prothrombin (1.4 microM) was added to homogeneously sized phospholipid vesicles of phosphatidylcholine-phosphatidylserine (3:1) at a total phospholipid concentration of 20 microM, the scattering intensity doubled. Upon subsequent addition of Factor Xa and Factor Va (5.0 nM each) the scattering intensity smoothly decreased to a value about 1.25-fold greater than that of the vesicles alone. Analysis of the composition of the reaction mixture at intervals during the course of the reaction by gel electrophoresis and laser densitometry, provided a good correlation between the mass of the vesicle-protein complex measured by light scattering and its mass inferred by composition. In addition, the decrease in mass of the vesicle-protein complex measured by light scattering correlated temporally with cleavage of Fragment 1.2. When the reaction was initiated in the presence of the reversible thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide no cleavage of Fragment 1.2 occurred, as indicated by gel electrophoresis, and no change in the mass of the vesicle-protein complex occurred as indicated by light scattering. The absence of change in scattering intensity in the presence of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide suggests a 1:1 replacement of prothrombin at the catalytic surface by components of equivalent mass (Fragment 1.2 plus thrombin), whereas the decrease in scattering in the absence of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide suggests replacement of prothrombin by Fragment 1 only. Together these results indicate that the thrombin-catalyzed cleavage of Fragment 1.2 promotes release of thrombin from the catalytic surface.     

Alpha-lactalbumin binding to membranes: evidence for a partially buried protein

The self-incorporation of apo-alpha-lactalbumin (alpha-LA) into single unilamellar vesicles (SUV) of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine was demonstrated by column chromatographic analyses on Sephadex G-200 (10 mM Tris-HCl, pH 7.4, 26-28 degrees C) and by intrinsic fluorescence emission of SUV-bound alpha-LA. It was shown that apo-alpha-LA slowly incorporated into the DMPC vesicle bilayer after equilibrating different mixtures of protein and SUV for several hours. The intrinsic fluorescence properties of the bound apo-alpha-LA were altered only slightly (lambda maxem = 333 nm vs. 337 nm in aqueous solution). The large blue shift in apo-alpha-LA fluorescence in solution induced by monovalent cations, such as Na(I), was almost completely prevented when apo-alpha-LA was membrane bound. Furthermore, the addition of calcium caused a slow conversion from apo-alpha-LA to Ca(II)-alpha-LA by a mechanism consistent with passive diffusion of Ca(II) into the bilayer interior to the (buried) calcium binding site. The release of Ca(II)-alpha-LA from the membrane is discussed with reference to alpha-LA release from the smooth endoplasmic reticulum in vivo.     

Passive Ca2+ fluxes across the membrane of sarcoplasmatic reticulum of skeletal muscles. The effect of calcium channel blockers

It was found that the initial rate of passive KC1-stimulated Ca2+ influx into sarcoplasmic reticulum (SR) vesicles follows the saturation kinetics at Ca2+ concentrations of 8-10 mM. The inhibitory effect of Ca2+ channel blockers (La3+, Mn2+, Co2+, Cd2+, Mg2+) on passive Ca2+ influx into SR vesicles is competitive with respect to Ca2+. These blockers also inhibit the initial fast phase of Ca2+ efflux from Ca2+-loaded SR vesicles. Verapamil (0.1-0.5 mM) added to the incubation mixture has no effect on passive Ca2+ fluxes across the SR vesicle membrane or on Ca2+ binding and ATP-dependent Ca2+ accumulation. However, preincubation of SR vesicles with verapamil (18 hours, 4 degrees C) or its introduction into the medium for SR vesicle isolation leads to the inhibition of passive Ca2+ fluxes.     

Neutron scattering determination of the binding of prothrombin to lipid vesicles

Low-angle neutron scattering is used to study the binding of human prothrombin to small single-bilayer vesicles consisting of phosphatidylcholine and phosphatidylserine (1/1 w/w). The radius of gyration of prothrombin indicates that it is an elongated molecule. The vesicles alone were not observed to coalesce, and their molecular weight, outer radius, and average surface area per lipid were respectively (1.6 +/- 0.32) X 10(6), 114 +/- 4 A, and 110 +/- 18 A2. These values were independent of the presence of calcium and were not altered significantly by prothrombin, which binds reversibly to the vesicle outer surface with its long axis projecting approximately radially forming a 90-A thick protein shell. From the titration of the protein-vesicle interaction, the apparent dissociation constant of the binding of prothrombin to these vesicles is estimated to be 0.8 +/- 0.4 microM. At saturation, 57 +/- 7 prothrombin molecules bind, giving 25 +/- 6 lipid residues and an area of 2900 +/- 400 A2 per prothrombin molecule on the vesicle outer surface. This area is about twice that calculated from a prolate ellipsoid model for prothrombin. However, it is close to the maximum cross-sectional area of fragment 1, the lipid binding region of prothrombin, which is coin-shaped in the high-resolution X-ray structure [Park, C.H., & Tulinsky, A. (1986) Biochemistry 25, 3977-3982]. This similarity suggests that prothrombin binding could be sterically limited.     

Interaction of the polyene antibiotic amphotericin B with model membranes: differences between small and large unilamellar vesicles

The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic and kinetic studies of the interaction of vesicular dipalmitoylphosphatidylcholine with Agkistrodon piscivorus piscivorus phospholipase A2

The tryptophan fluorescence emission intensity at 340 nm of monomeric phospholipase A2 from Agkistrodon piscivorus piscivorus increased about 70% upon addition of dipalmitoylphosphatidylcholine small unilamellar vesicles (DPPC SUV) at 25 degrees C. The emission spectrum was also blue-shifted 6-8 nm, suggesting that the environment of 1 or more tryptophan residues had become less polar. This effect of SUV on the phospholipase A2 fluorescence was independent of Ca2+ at 25 degrees C, and the apparent association constant for the interaction was approximately 1.7 x 10(4) M-1. The apparent Km for hydrolysis of DPPC SUV was equal to the inverse of the estimated association constant. In the absence of Ca2+, the change in fluorescence intensity decreased with increasing temperature. Thermodynamic analysis of this reversible, temperature-dependent fluorescence change indicated that the A. p. piscivorus monomer phospholipase A2 interacts only with SUV in the true gel phase existing below the pretransition of gel to "ripple" phase lipid in the absence of Ca2+. In contrast, the fluorescence intensity change upon addition of SUV in the presence of Ca2+ was independent of temperature over the range of 25-48 degrees C. Under these conditions, hydrolysis of the lipid occurred concomitantly with the change in fluorescence which could not be reversed by the addition of EDTA. With a nonhydrolyzable analog of DPPC, however, the fluorescence changes upon mixing of SUV, Ca2+, and phospholipase A2 were reversible and temperature-dependent. Thus, the apparent irreversibility of the change in fluorescence observed with Ca2+ and DPPC SUV was correlated with hydrolysis of the vesicles. These results indicate that the magnitude of the initial interaction of enzyme with substrate is reversible, is Ca2+-independent, depends upon the lipid state, and is quantitatively correlated to the maximum rate of hydrolysis.     

Modulation of membrane fusion by calcium-binding proteins.

The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion.     

Exchange and flip-flop of dimyristoylphosphatidylcholine in liquid-crystalline, gel, and two-component, two-phase large unilamellar vesicles

The rate and extent of spontaneous exchange of dimyristoylphosphatidylcholine (DMPC) from large unilamellar vesicles (LUV) composed of either DMPC or mixtures of DMPC/distearoylphosphatidylcholine (DSPC) have been examined under equilibrium conditions. The phase state of the vesicles ranged from all-liquid-crystalline through mixed gel/liquid-crystalline to all-gel. The exchange rate of DMPC between liquid-crystalline DMPC LUV, measured between 25 and 55 degrees C, was found to have an Arrhenius activation energy of 24.9 +/- 1.4 kcal/mol. This activation energy and the exchange rates are very similar to those obtained for the exchange of DMPC between DMPC small unilamellar vesicles (SUV). The extent of exchange of DMPC in LUV was found to be approximately 90%. This is in direct contrast to the situation in DMPC SUV where only the lipid in the outer monolayer is available for exchange. Thus, transbilayer movement (flip-flop) is substantially faster in liquid-crystalline DMPC LUV than in SUV. Desorption from gel-phase LUV has a much lower rate than gel-phase SUV with an activation energy of 31.7 +/- 3.7 kcal/mol compared to 11.5 +/- 2 kcal/mol reported for SUV. A defect-mediated exchange in gel-phase SUV, which is not the major pathway for exchange in LUV, is proposed on the basis of the thermodynamic parameters of the activation process. Surprisingly, the rates of DMPC exchange between DMPC/DSPC two-component LUV, measured over a wide range of compositions and temperatures, were found to exhibit very little dependence on the composition or phase configuration of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.     

Evidence for self-association of prothrombin fragment 1 in the absence of calcium ions. Implications for the interpretation of cooperativity of calcium binding

Sedimentation equilibrium studies have demonstrated that prothrombin fragment 1 from either human or bovine plasma reversibly dimerizes in the absence of Ca2+ with an equilibrium constant of 1,000 M-1. In the presence of 10 mM Ca2+ this association constant increased to 10,000 M-1. A model for preferential binding of Ca2+ to the pre-existing dimer has been found capable of accounting quantitatively for the cooperative Ca2+ binding to this prothrombin fragment, and for the dependence of its sedimentation coefficient on protein concentration in the presence and absence of metal ion. Sedimentation equilibrium studies of intact bovine and human prothrombins have confirmed previous reports that these prothrombins dimerize. For both prothrombins the association constant is 10,000 M-1, both in the absence and presence of Ca2+.     

Interaction of human thrombin I fragment, prethrombin I, and alpha-thrombin with tissue thromboplastin]

The binding of 125I-labeled prothrombin fragment I. prethrombin I and alpha-thrombin to native and papain-treated tissue thromboplastin in the presence of CaCl2 of EDTA was studied. The experimental curves plotted in the Scatchard coordinates testify to the presence in thromboplastin of two types of fragment I binding sites: those with a high (Kd = 7.6 x 10(-6) M) and moderate (Kd = 1.3 x 10(-8) M) binding affinity. The parameters of fragment I binding and their changes reproduced, for the most part, the mode of prothrombin binding observed in previous studies. The experimental results provide indirect evidence in favour of a hydrophobic role of Ca(2+)-dependent binding of prothrombin fragment I to thromboplastin. The binding of prethrombin I was nonspecific and Ca(2+)-independent, whereas alpha-thrombin showed a relatively high level of nonspecific electrostatic binding which was competitively inhibited by Ca2+. Thromboplastin proteins interacted (both directly and in a Ca(2+)-independent fashion) with all the prothrombin derivatives under study.