Dioleoylphosphatidylserine Research Articles

Structural basis for autoantibody recognition of phosphatidylserine-beta 2 glycoprotein I and apoptotic cells.

Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by beta 2 glycoprotein I (beta 2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-beta 2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells.

Alamethicin channel conductance modified by lipid charge.

The membrane surface charge modifies the conductance of ion channels by changing the electric potential and redistributing the ionic composition in their vicinity. We have studied the effects of lipid charge on the conductance of a multi-state channel formed in planar lipid bilayers by the peptide antibiotic alamethicin. The channel conductance was measured in two lipids: in a neutral dioleoylphosphatidylethanolamine (DOPE) and a negatively charged dioleoylphosphatidylserine (DOPS). The charge state of DOPS was manipulated by the pH of the membrane-bathing solution. We find that at high salt concentrations (e.g., 2 M NaCl) the effect of the lipid charge is below the accuracy of our measurements. However, when the salt concentration in the membrane-bathing solution is decreased, the surface charge manifests itself as an increase in the conductance of the first two channel levels that correspond to the smallest conductive alamethicin aggregates. Our analysis shows that both the salt and pH dependence of the surface charge effect can be rationalized within the nonlinear Poisson-Boltzmann approach. Given channel conductance in neutral lipids, we use different procedures to account for the surface charge (e.g., introduce averaging over the channel aperture and take into account Na+ adsorption to DOPS heads), but only one adjustable parameter: an effective distance from the nearest lipid charge to the channel mouth center. We show that this distance varies by 0.3-0.4 nm upon channel transition from the minimal conducting aggregate (level L0) to the next larger one (level L1). This conclusion is in accord with a simple geometrical model of alamethicin aggregation.

Influence of lipid composition on physical properties and peg-mediated fusion of curved and uncurved model membrane vesicles: "nature's own" fusogenic lipid bilayer.

Poly(ethylene glycol) (PEG)-mediated fusion of phosphatidylcholine model membranes has been shown to mimic the protein-mediated biomembrane process [Lee, J., and Lentz, B. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9274-9279]. Unlike the simple model membranes used in this earlier study, the lipid composition of fusogenic biomembranes is quite complex. The purpose of this paper was to examine PEG-mediated fusion of highly curved (SUV) and largely uncurved (LUV) membrane vesicles composed of different lipids in order to identify lipid compositions that produce highly fusogenic membranes. Starting with liposomes composed of five lipids with different physical properties, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), bovine brain sphingomyelin (SM), and cholesterol (CH), we systematically varied the composition and tested for the extent of PEG-mediated fusion after 5 min of treatment. We found that a vesicle system composed of four lipids, DOPC/DOPE/SM/CH, fused optimally at a 35/30/15/20 molar ratio. Each lipid seemed to play a part in optimizing the membrane for fusion. PE disrupted outer leaflet packing as demonstrated with TMA-DPH lifetime, C(6)-NBD-PC partitioning, and DPH anisotropy measurements, and thus significantly enhanced fusion and rupture, without significantly altering interbilayer approach (X-ray diffraction). An optimal ratio of PC/PE (35/30) produced a balance between fusion and rupture. CH and SM, when present at an optimal ratio of 3/4 in vesicles containing the optimal PC/PE ratio, reduced rupture without significantly reducing fusion. This optimal CH/SM ratio also enhanced outer leaflet packing, suggesting that fusion is dependent not only on outer leaflet packing but also on the properties of the inner leaflet. Addition of CH without SM enhanced rupture relative to fusion, while SM alone reduced both rupture and fusion. The optimal lipid composition is very close to the natural synaptic vesicle composition, suggesting that the synaptic vesicle composition is optimized with respect to fusogenicity.

Two-Dimensional Crystallization of Annexin A5 on Phospholipid Bilayers and Monolayers: A Solid−Solid Phase Transition between Crystal Forms

Annexin A5 is a soluble protein which binds to negatively charged phospholipids, such as dioleoylphosphatidylserine (DOPS), in the presence of Ca2+. A solid−solid phase transition between two two-dimensional crystal forms of annexin A5, p6 and p3, previously identified by electron microscopy (EM), was studied in situ on supported phospholipid bilayers (SPBs) by atomic force microscopy (AFM) and ex situ on lipid monolayers by EM. The ability to directly vary the protein surface density on SPBs allowed a delicate and systematic way to investigate the reversibility of the transition with the AFM. The p6 crystal form was found to form first and to cover the entire lipid surface available, at all DOPS concentrations studied (5−95%). An increase in protein surface density, achievable only on SPBs (or monolayers) containing sufficient amounts of DOPS, destabilized the p6 phase. The transition to the more compact (p3) form occurred via local melting of the p6 phase at defects, such as grain boundaries, initially present in p6 crystals. On mono- or bilayers with lower DOPS contents, the density-dependent p6−p3 transition was not observed, but the p3 crystal form could still be reached by disturbing the system mechanically.

Dipole Potentials of Monolayers of Phosphatidylcholine, Phosphatidylserine, and Phosphatidic Acid on Mercury

A novel procedure for the measurement of the absolute value of the surface dipole potential χ of self-assembled lipid monolayers, which makes use of a phospholipid monolayer supported on mercury, is described. It consists of increasing the tilt angle of the lipid molecules with respect to the monolayer normal by expanding progressively the supporting mercury drop and in measuring the charge on the mercury surface that accompanies the drop expansion. Dipole potential values of +145 ± 10 mV for neutral dioleoylphosphatidylcoline (DOPC) and dioleoylphosphatidylserine (DOPS) and of +30 ± 3 mV for neutral dioleoylphosphatidic acid (DOPA) were determined. A gradual increase in the negative charge of the DOPS and DOPA headgroups due to an increase in pH causes an increase in χ. This suggests that the dipole potential of about +145 mV in neutral dioleoylphospholipids stems from the ester linkages to the glycerol backbone (which in DOPA monolayers are screened by the water molecules) and possibly, to a minor extent, from the orientation of the hydration water molecules. The latter contribution is small in neutral phospholipid monolayers, but becomes progressively more positive with an increase in the negative charge on the polar heads of the lipids.

Effect of Ca2+ on the Morphology of Mixed DPPC−DOPS Supported Phospholipid Bilayers

The morphology of supported phospholipid bilayers (SPBs) containing mixtures of phospholipids in gel (dipalmitoyl phosphatidylcholine, DPPC) and fluid (dioleoyl phosphatidylserine (DOPS) or -choline (DOPC)) states at room temperature was investigated by atomic force microscopy (AFM). Fluid−gel phase separation was clearly detectable on topography images of mixed SPBs. The presence or absence of Ca2+ in the subphase was found to have a dramatic effect on the organization of the gel phase when the fluid phase was composed of pure DOPS: In the absence of Ca2+, large, well-defined DPPC domains were found in both the DPPC/DOPC and DPPC/DOPS mixtures, while in its presence small, isolated DPPC domains were found in the DPPC/DOPS mixture. Ca2+ had no effect on the organization of DPPC in DPPC/DOPC mixtures, and its effect was abolished by adding DOPC to DPPC/DOPS mixtures.

Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

Accumulation of Ca(2+) by the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum has been measured in reconstituted, sealed vesicles as a function of lipid composition. Measurements were performed in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to eliminate any effects of H(+) transport; in the presence of FCCP, addition of valinomycin had no effect on the level or rate of accumulation of Ca(2+) showing that, in the presence of FCCP, no electrical potential built up across the membrane. Levels of accumulation were low when the phospholipid was dioleoylphosphatidylcholine (DOPC), even though DOPC supports high ATPase activity. Inclusion of 10 mol% anionic phospholipid [dioleoylphosphatidic acid (DOPA) or dioleoylphosphatidylserine (DOPS)] led to higher levels of accumulation of Ca(2+), 10 mol% being the optimum concentration. Cardiolipin or phosphatidylinositol 4-phosphate were more effective than DOPA or DOPS in increasing accumulation of Ca(2+). Effects of anionic phospholipids were seen in the presence of an ATP-regenerating system to remove ADP, and in the presence of phosphate within the reconstituted vesicles to precipitate calcium phosphate. Rates of passive leak of Ca(2+) from the reconstituted vesicles were slow. The Ca(2+)-accumulation process was simulated assuming either simple passive leak of Ca(2+) from the vesicles or assuming slippage on the ATPase, a process in which the phosphorylated intermediate of the ATPase releases bound Ca(2+) on the cytoplasmic rather than the lumenal side of the membrane. The experimental data fitted to a slippage model, with anionic phospholipids decreasing the rate of slippage.

Force Measurements on Myelin Basic Protein Adsorbed to Mica and Lipid Bilayer Surfaces Done with the Atomic Force Microscope

The mechanical and adhesion properties of myelin basic protein (MBP) are important for its function, namely the compaction of the myelin sheath. To get more information about these properties we used atomic force microscopy to study tip-sample interaction of mica and mixed dioleoylphosphatidylserine (DOPS) (20%)/egg phosphatidylcholine (EPC) (80%) lipid bilayer surfaces in the absence and presence of bovine MBP. On mica or DOPS/EPC bilayers a short-range repulsive force (decay length 1.0–1.3 nm) was observed during the approach. The presence of MBP always led to an attractive force between tip and sample. When retracting the tip again, force curves on mica and on lipid layers were different. While attached to the mica surface, the MBP molecules exhibited elastic stretching behavior that agreed with the worm-like chain model, yielding a persistence length of 0.5 ± 0.25 nm and an average contour length of 53 ± 19 nm. MBP attached to a lipid bilayer did not show elastic stretching behavior. This shows that the protein adopts a different conformation when in contact with lipids. The lipid bilayer is strongly modified by MBP attachment, indicating formation of MBP-lipid complexes and possibly disruption of the original bilayer structure.

Lipid packing stress and polypeptide aggregation: alamethicin channel probed by proton titration of lipid charge.

Lipid membranes are not passive, neutral scaffolds to hold membrane proteins. In order to examine the influence of lipid packing energetics on ion channel expression, we study the relative probabilities of alamethicin channel formation in dioleoylphosphatidylserine (DOPS) bilayers as a function of pH. The rationale for this strategy is our earlier finding that the higher-conductance states, corresponding to larger polypeptide aggregates, are more likely to occur in the presence of lipids prone to hexagonal HII-phase formation (specifically DOPE), than in the presence of lamellar L alpha-forming lipids (DOPC). In low ionic strength NaCl solutions at neutral pH, the open channel in DOPS membranes spends most of its time in states of lower conductance and resembles alamethicin channels in DOPC; at lower pH, where the lipid polar groups are neutralized, the channel probability distribution resembles that in DOPE. X-Ray diffraction studies on DOPS show a progressive decrease in the intrinsic curvature of the constituent monolayers as well as a decreased probability of HII-phase formation when the charged lipid fraction is increased. We explore how proton titration of DOPS affects lipid packing energetics, and how these energetics couple titration to channel formation.

Characterization of the Growth of 2D Protein Crystals on a Lipid Monolayer by Ellipsometry and Rigidity Measurements Coupled to Electron Microscopy

We present here some sensitive optical and mechanical experiments for monitoring the process of formation and growth of two-dimensional (2D) crystals of proteins on a lipid monolayer at an air-water interface. The adsorption of proteins on the lipid monolayer was monitored by ellipsometry measurements. An instrument was developed to measure the shear elastic constant (in plane rigidity) of the monolayer. These experiments have been done using cholera toxin B subunit (CTB) and annexin V as model proteins interacting with a monosialoganglioside (GM1) and dioleoylphosphatidylserine (DOPS), respectively. Electron microscopy observations of the protein-lipid layer transferred to grids were systematically used as a control. We found a good correlation between the measured in-plane rigidity of the monolayer and the presence of large crystalline domains observed by electron microscopy grids. Our interpretation of these data is that the crystallization process of proteins on a lipid monolayer passes through at least three successive stages: 1) molecular recognition between protein and lipid-ligand, i.e., adsorption of the protein on the lipid layer; 2) nucleation and growth of crystalline patches whose percolation is detected by the appearance of a non-zero in-plane rigidity; and 3) annealing of the layer producing a slower increase of the lateral or in-plane rigidity.

Adsorption and reduction kinetics of safranine T in self-assembled phospholipid monolayers deposited on mercury

Abstract The adsorption of safranine-T (SFR) in self-assembled monolayers of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidic acid (DOPA) deposited on a hanging mercury drop electrode was measured over the concentration range from 1 × 10 −7 to 2 × 10 −5 M, and its electroreduction was investigated over the pH range from 2 to 6 by a computerized chronocoulometric technique. The mechanism of SFR electroreduction on a DOPC-coated mercury electrode was elucidated on the basis of the dependence of the applied potential upon the electrolysis time t at constant pH and upon pH at constant t , while keeping the faradaic charge constant: the SFR cation is electroreduced in a two-electron process which takes place via the reversible uptake of the first transferring electron with formation of the neutral radical SFR, followed by the rate-determining protonation of this radical with SFRH .+ formation. On DOPA and DOPS monolayers the electroreduction rate of SFR is higher than on DOPC monolayers, possibly due to an acceleration of the protonation step by the protonated phosphate groups of the polar heads of these phospholipids.

Interaction of poly(ethylene-glycols) with air-water interfaces and lipid monolayers: investigations on surface pressure and surface potential

We have characterized the surface activity of different-sized poly(ethylene-glycols) (PEG; M(r) 200-100,000 Da) in the presence or absence of lipid monolayers and over a wide range of bulk PEG concentrations (10(-8)-10% w/v). Measurements of the surface potential and surface pressure demonstrate that PEGs interact with the air-water and lipid-water interfaces. Without lipid, PEG added either to the subphase or to the air-water interface forms relatively stable monolayers. Except for very low molecular weight polymers (PEGs < 1000 Da), low concentrations of PEG in the subphase (between 10(-5) and 10(-4)% w/v) increase the surface potential from zero (with respect to the potential of a pure air-water interface) to a plateau value of approximately 440 mV. At much higher polymer concentrations, > 10(-1)% (w/v), depending on the molecular weight of the PEG and corresponding to the concentration at which the polymers in solution are likely to overlap, the surface potential decreases. High concentrations of PEG in the subphase cause a similar decrease in the surface potential of densely packed lipid monolayers spread from either diphytanoyl phosphatidylcholine (DPhPC), dipalmitoyl phosphatidylcholine (DPPC), or dioleoyl phosphatidylserine (DOPS). Adding PEG as a monolayer at the air-water interface also affects the surface activity of DPhPC or DPPC monolayers. At low lipid concentration, the surface pressure and potential are determined by the polymer. For intermediate lipid concentrations, the surface pressure-area and surface potential-area isotherms show that the effects due to lipid and PEG are not always additive and that the polymer's effect is distinct for the two lipids. When PEG-lipid-mixed monolayers are compressed to surface pressures greater than the collapse pressure for a PEG monolayer, the surface pressure-area and surface potential-area isotherms approach that of the lipid alone, suggesting that for this experimental condition PEG is expelled from the interface.

Production of thrombin as a probe for mixing of phospholipids in membranes on solid supports

Phospholipid-covered solid supports have been used successfully as model membranes in studies on blood coagulation and other research fields. In order to produce such membranes, simple exposure of the support to suspensions of phospholipid vesicles was recently introduced, but questions have remained about the process of vesicle adherence to the surface and the physico-chemical properties of the resulting membranes. Using a new technique, mixing of phospholipids in such membranes was demonstrated. A rotating, hydrophilic, silicon disc was exposed in a two-step procedure to vesicles prepared from mixtures of dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylcholine (DOPC). Factor Xa, factor Va and prothrombin were added and the transport-limited production rate of thrombin was measured. For low surface coverage with 40% DOPS/60% DOPC, a much higher conversion rate was found if, prior to addition of coagulation factors, excess DOPC vesicles were added to fill up vacant surface area. It is concluded that DOPS is spread over the entire surface and that confluent bilayers are formed. The presented technique may also be used to measure lateral diffusion constants.

Enhanced hydrolysis of phosphatidylcholine by human group II non-pancreatic secreted phospholipase A2 as a result of interfacial activation by specific anions. Potential role of cholesterol sulphate.

The extracellular concentration of the Group II human non-pancreatic secreted phospholipase A2 (hnpsPLA2) is elevated in a variety of inflammatory disorders. This enzyme is remarkable because it demonstrates almost zero activity with egg phosphatidylcholine (PC) or synthetic dioleoyl-phosphatidylcholine (DOPC) as substrate, but expresses high activity with the anionic phospholipid dioleoyl-phosphatidylglycerol (DOPG), a feature shared with the Group II enzyme from rat liver. The presence of certain membrane-bound anions can enhance hydrolysis of PC by the mammalian secreted PLA2S. In this study the ability of various non-polar anions to stimulate DOPC hydrolysis by secreted PLA2S has been investigated. The naturally occurring membrane anion, cholesterol sulphate, was particularly effective in stimulating the hydrolysis of both DOPC and also 1-stearoyl-2-arachidonyl phosphatidylcholine by hnpsPLA2. Activation of DOPC hydrolysis was also achieved with dioleoyl-phosphatidylserine (DOPS); however, DOPS was less effective than cholesterol sulphate. In contrast, the dianion dioleoyl-phosphatidic acid, a known activator of pig pancreatic PLA2, failed to activate the human enzyme. It remains to be established whether cell plasma-membrane hydrolysis by extracellular hnpsPLA2 can be activated in vivo by the presence of suitable membrane anions such as cholesterol sulphate and thus promote an inflammatory response within the cell.

Effect of synthetic phospholipids on the response of the activated partial thromboplastin time to heparin

The activated partial thromboplastin time (aPTT) is the most popular test for monitoring of heparin therapy. The purpose of the present study was to show that an aPTT reagent with good response to heparin can be prepared from synthetic phosphoglycerides. Mixed liposomes were prepared from synthetic dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylcholine (DOPC), and dioleoylphosphatidylethanolamine (DOPE). These liposomes were used in an aPTT test system with kaolin as activator, to evaluate their procoagulant activity in the absence and presence of heparin. For comparison, mixtures of purified non-synthetic phospholipids were prepared and tested with the same systems. The aPTT and its response to heparin were influenced by the phospholipid class composition and concentration. The presence of phosphatidylserine (PS) was required to reduce the aPTT of normal plasma to values between 30 and 40s. The presence of phosphatidylethanolamine (PE) in mixed liposomes could modulate the response to heparin. At low PE/PS liposome concentrations (approximately 40 microM), a relatively low response was observed. At high liposome concentrations (approximately 1 mM), the response to heparin increased with the mole fraction of phosphatidylethanolamine. The results obtained with non-synthetic phospholipid mixtures were similar to those obtained with the synthetic phosphoglycerides. Optimal concentrations of DOPS, DOPE and DOPC were found with which an almost linear response to heparin and to low molecular weight heparin (Fragmin) was observed. Using a mixed liposome consisting of 12 microM DOPS/12 microM DOPC/16 microM DOPE, a doubling of the base-line aPTT was achieved at approximately 0.2 IU/ml of heparin, and at approximately 1.0 IU/ml of Fragmin.(ABSTRACT TRUNCATED AT 250 WORDS)

Testing protein adsorption models by off-null ellipsometry: Determination of binding constants from a single adsorption curve

The kinetics of prothrombin adsorption on planar bilayers, prepared from 40% dioleoyl phosphatidylserine and 60% dioleoyl phosphatidylcholine, are studied by off-null ellipsometry. A simple expression for the adsorption rate is derived directly from measured adsorption rates. The interpretation of this expression in terms of transport processes shows that up to a surface coverage Γ = 1.8 pmol cm −2, i.e. 50% of the maximal surface coverage Γ max, the adsorption rate appears to be regulated by diffusion-limited transport of prothrombin from the bulk solution to the adsorbing surface. The adsorption rate is proportional to the concentration difference between the bulk and the solution immediately adjacent to the surface. The latter concentration appears to be instantaneously in equilibrium with the surface excess, conforming to the classical Langmuir isotherm. For low surface coverages, below 50% of maximal adsorption, the kinetics are thus described by a simple model containing the mass transfer coefficient, the dissociation constant K d and the maximal surface coverage Γ max, With known Γ max this model allows the estimation of K d from a single adsorption curve. However, for surface coverages exceeding 60% of maximal adsorption, drastic deviations from this model are found. The intrinsic adsorption rate is substantially reduced, presumably by surface exclusion.

Monitoring of unbound protein in vesicle suspensions with off-null ellipsometry

In studies on the binding of proteins to small unilamellar phospholipid vesicles (SUV), the concentration of unbound protein usually remains unknown, because the vesicles cannot be separated from the bulk solution. In the present study, this limitation was overcome by addition of a supported planar phospholipid bilayer to the cuvette containing a vesicle suspension. Ellipsometric measurement of the protein adsorption velocities on this bilayer allowed determination of the concentrations of unbound protein. At high protein concentrations the adsorption is rapidly completed and the usual null-ellipsometry is too slow to obtain well-defined initial adsorption rates. Therefore, an off-null technique was developed, allowing measurement of the adsorbed protein mass at time intervals of 20 ms. Binding of prothrombin and coagulation factor Xa was measured in SUV suspensions prepared from a 20% dioleoylphosphatidylserine (DOPS) and 80% dioleoylphosphatidylcholine (DOPC) phospholipid mixture. For prothrombin, a dissociation constant K d = 140 ± 27 nM (mean ± S.E.) and maximal surface concentration Γ max = (8.9 ± 0.8) · 10 −3 mole of protein per mole of lipid, were obtained. For factor Xa, these values were K d = 49.6 ± 6.3 nM and Γ max = (23.0 ± 1.4) · 10 −3 mole of protein per mole of lipid. These binding parameters are similar to those obtained earlier for planar bilayers. Apparently, the binding of factor Xa and prothrombin is not dependent on surface curvature.

Membrane-mediated assembly of the prothrombinase complex.

Prothrombinase assembly was studied on macroscopic planar bilayers consisting of 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC). The dissociation constant for the binding of factor Xa to the bilayer, measured by ellipsometry, was Kd = 47 +/- 8 nM (mean +/- S.D.) and this value was lowered to Kd = 2.2 +/- 0.3 pM by preadsorption of factor Va. This latter value was determined from direct measurement of steady-state thrombin production. A comparable value of Kd = 1.0 +/- 0.1 pM was found by repeating these experiments in suspensions of phospholipid vesicles, and it was verified that prothrombinase assembly was not influenced by the addition of prothrombin. Using a minute amount (0.094 fmol cm-2) of preadsorbed factor Va, it was found that the rate of prothrombinase assembly exceeds the rate of collisions between Xa molecules from the buffer and the sparse Va molecules on the bilayer. Apparently, factor Xa adsorbs first to the membrane and then associates rapidly with factor Va by lateral diffusion. The data indicate almost instantaneous equilibrium of this complex formation on the surface with a lower limit for the bimolecular rate constant of kon = 2.8 x 10(13) (mol/cm2)-1 s-1. In suspensions of small phospholipid vesicles, prothrombinase assembly is collisionally limited and the value of kon should be proportional to vesicle diameter. This was verified with a method for estimation of kon values from thrombin generation curves. Values of 0.36 x 10(9) and 1.6 x 10(9) M-1 s-1 were found for vesicles of 20-30- and 60-80-nm diameter, respectively.

Comparable interaction of doxorubicin with various acidic phospholipids results in changes of lipid order and dynamics

We have characterized the interaction of the antitumor drug doxorubicin woth model membranes of the anionic phospholipids dioleoylphospatidic acid (DOPA), dioleoylphosphatidylserine (DOPS), cardiolipin and dioleoylphosphatidylglycerol (DOPG) as compared to the zwitterionic dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE). The saturating binding levels were: 2.4 (DOPA), 1.3 (cardiolipin), 1.5 (DOPS, DOPG) and 0.02 (DOPC) doxorubicin per lipid phosphorus (mol/mol). The half-saturating free drug concentrations were comparable for DOPA, cardiolipin, DOPS and DOPG: 20, 16, 35 and 18 μM, respectively. Doxorubicin fluorescence revealed the simultaneous existence of at least two populations of bound drug in the various anionic phospholipids: (1) fluorescent molecules with chromophores that reside between the lipid molecules and (2) above 0.01–0.02 doxorubicin bound per lipid phosphorus: non-fluorescent drug-stacks that are closer to the aqueous phase than the fluorescent molecules. Small-angle X-ray scattering indicated that doxorubicin can reorganize anionic phospholipid dispersions into closely-packed multilamellar structures. Addition of the drug caused leakage of entrapped 6-carboxyfluorescein. Neither 2H-NMR on [2- 2H]serine-labelled DOPS nor 31P-NMR revealed any significant effect of doxorubicin on headgroup conformation, but 2H-NMR on di[11,11- 2H 2]oleoyl-labelled phospholipids showed that the drug had a strong acyl chain-disordering effect on anionic phospholipids. 2H-NMR relaxation measurements indicated that the drug immobilized the headgroups and acyl chains of anionic phospholipids. The implications of these observations for the cellular activity of the drug are indicated.

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.