Nonideal mixing of phosphatidylserine and phosphatidylcholine in the fluid lamellar phase.

The mixing of phosphatidylserine (PS) and phosphatidylcholine (PC) in fluid bilayer model membranes was studied by measuring binding of aqueous Ca2+ ions. The measured [Ca2+]aq was used to derive the activity coefficient for PS, gamma PS, in the lipid mixture. For (16:0, 18:1) PS in binary mixtures with either (16:0, 18:1)PC, (14:1, 14:1)PC, or (18:1, 18:1)PC, gamma PS > 1; i.e., mixing is nonideal, with PS and PC clustered rather than randomly distributed, despite the electrostatic repulsion between PS headgroups. To understand better this mixing behavior, Monte Carlo simulations of the PS/PC distributions were performed, using Kawasaki relaxation. The excess energy was divided into an electrostatic term Uel and one adjustable term including all other nonideal energy contributions, delta Em. Uel was calculated using a discrete charge theory. Kirkwood's coupling parameter method was used to calculate the excess free energy of mixing, delta GEmix, hence In gamma PS,calc. The values of In gamma PS,calc were equalized by adjusting delta Em in order to find the simulated PS/PC distribution that corresponded to the experimental results. We were thus able to compare the smeared charge calculation of [Ca2+]surf with a calculation ("masked evaluation method") that recognized clustering of the negatively charged PS: clustering was found to have a modest effect on [Ca2+]surf, relative to the smeared charge model. Even though both PS and PC tend to cluster, the long-range nature of the electrostatic repulsion reduces the extent of PS clustering at low PS mole fraction compared to PC clustering at an equivalent low PC mole fraction.     

Proton-induced phase separation in phosphatidylserine/phosphatidylcholine membranes

Effects of ph and ionic strength on phosphatidylserine/phosphatidylcholine mixed membranes prepared on Millipore filter pore surfaces have been studied using spin-labeled phosphatidylcholine. Lowering pH at constant ionic strength and lowering ionic strength at constant pH caused a lateral reorganization of the membrane. The trigger was protonation of the serine carboxyl group which caused solidification of phosphatidylserine molecules in the membrane, leaving a fluid phase consisting mainly of phosphatidylcholine. The appearent pK for the proton-induced phase separation was measured in a wide range of salt concentrations. The ionic strength dependence was satisfactorily explained based on the electrostatic free energy of proton in the field of membrane surface potential. The Gouy-Chapman theory gave a good approximation for the surface potential. The surface pK of phosphatidylserine and phosphatidic acid vesicles was directly measured in various salt concentrations by 31P-NMR and the results confirmed validity of the Gouy-Chapman-type analysis. The lateral reorganization was triggered by electrostatic interaction but the bulk of the stabilization energy for the structural changes would be the gains in intermolecular van der Waals energy due to closer packing of phosphatidylserine on solidification.     

Effects of diacylglycerols on conformation of phosphatidylcholine headgroups in phosphatidylcholine/phosphatidylserine bilayers.

The effects of five diacylglycerols (DAGs), diolein, 1-stearoyl,2-arachidonoyl-sn-glycerol, dioctanoylglycerol, 1-oleoyl,2-sn-acetylglycerol, and dipalmitin (DP), on the structure of lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine (4:1 mol/mol) were examined by 2H nuclear magnetic resonance (NMR). Dipalmitoylphosphatidylcholine deuterated at the alpha- and beta-positions of the choline moiety was used to probe the surface region of the membranes. Addition of each DAG except DP caused a continuous decrease in the beta-deuteron quadrupole splittings and a concomitant increase in the alpha-deuteron splittings indicating that DAGs induce a conformational change in the phosphatidylcholine headgroup. Additional evidence of conformational change was found at high DAG concentrations (> or = 20 mol%) where the alpha-deuteron peaks became doublets indicating that the two alpha-deuterons were not equivalent. The changes induced by DP were consistent with the lateral phase separation of the bilayers into gel-like and fluid-like domains with the phosphatidylcholine headgroups in the latter phase being virtually unaffected by DP. The DAG-induced changes in alpha-deuteron splittings were found to correlate with DAG-enhanced protein kinase C (PK-C) activity, suggesting that the DAG-induced conformational changes of the phosphatidylcholine headgroups are either directly or indirectly related to a mechanism of PK-C activation. 2H NMR relaxation measurements showed significant increase of the spin-lattice relaxation times for the region of the phosphatidylcholine headgroups, induced by all DAGs except DP. However, this effect of DAGs did not correlate with the DAG-induced activation of PK-C.     

Calcium ion binding between lipid bilayers: the four-component system of phosphatidylserine, phosphatidylcholine, calcium chloride, and water

Ca2+ binding between lamellae of phosphatidylserine (PS) and phosphatidylcholine (PC) gives rise to a rigid phase of Ca(PS)2. When aqueous Ca2+, hydrated PS/PC, and Ca(PS)2 coexist at equilibrium, the aqueous Ca2+ concentration is invariant and is characteristic of the PS/PC ratio. This characteristic Ca2+ concentration is 0.040 microM for palmitoyloleoylphosphatidylserine without PC and increases as the inverse square of the PS mole fraction at high PS concentration (Raoult's law) and as the inverse square of the PS mole fraction multiplied by a constant at low PS concentration (Henry's law). For example, for palmitoyloleoylphosphatidylserine/palmitoyloleoylphosphatidylcholi ne = 0.6/0.4 or 0.2/0.8, this characteristic Ca2+ concentration is about 0.1 or about 6 microM, respectively. These observations at constant temperature are summarized in a quaternary phase diagram for the four-component system CaCl2/PS/PC/water.     

Clustering of lecithin molecules in phosphatidylserine membranes induced by calcium ion binding to phosphatidylserine

The effects of calcium ion on phosphatidyl L-serine (PS) have been studied with PS membranes containing a lecithin spin label (L¹). The calcium ion makes the ESR spectra of the L¹ in PS membranes broadened owing to the intermolecular spin-spin exchange interactions. The results indicate that the calcium ion binds to PS molecules to form rapidly rigid calcium ion-bound PS aggregates, the lecithin molecules being thereby separated from the host PS bilayers to form clusters. The magnesium ion is ineffective for the aggregation and exerts a quite different effect only at much higher concentrations.     

On the nature of calcium ion binding between phosphatidylserine lamellae

Ca2+ binding between phosphatidylserine (PS) lamellae gives rise to a phase with the composition Ca(PS)2. When aqueous Ca2+, hydrated PS, and Ca(PS)2 coexist at equilibrium, the aqueous Ca2+ concentration is invariant. At Ca2+ concentrations below this critical value, no binding of Ca2+ to PS is detected. Above this value, Ca2+ binds to PS to form Ca(PS)2. The invariant Ca2+ concentration is 0.14 microM for palmitoyloleoylphosphatidylserine (POPS) and 3.0 microM for dioleoylphosphatidylserine (DOPS). For the mixed acyl chain PS derived from bovine brain (BBPS) this Ca2+ concentration ranges from 0.25 to 0.7 microM. The observed phase behavior is described by the phase rule for the three-component system of water, Ca2+, and PS, with temperature and pressure constant. In order for Ca2+ to bind between PS lamellae to form the Ca(PS)2 phase, the aqueous Ca2+ concentration must be supersaturated. The equilibrium Ca2+ concentration is determined by dissolving Ca(PS)2 by use of Ca2+ chelators.     

Detection of phase separation in fluid phosphatidylserine/phosphatidylcholine mixtures.

The nonideal mixing of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine, (16:0, 18:1)PS, and 1,2-didodecenoyl-sn-glycero-3-phosphocholine, (12:1, 12:1)PC, in fluid lamellar model membranes was studied by measuring binding of aqueous Ca2+ ions and by x-ray diffraction. A region of two-phase coexistence was found by invariance of the aqueous concentration and by the appearance of two sets of lamellar spacings. The phases were identified as fluid from the diffuse x-ray diffraction in the wide-angle region. The width of the two-phase coexistence region was greater at higher ionic strength. In 800 mM KCl, the phase boundaries were at PS mole fraction 0.5 and 0.8. In 100 mM KCl, the phase boundaries were at PS mole fraction 0.52 and 0.62. Monte Carlo simulations of the lateral distributions of these PS/PC mixtures show pronounced clustering of the lipids.     

Calcium binding protein from porcine intestine binds to phosphatidylserine vesicles in the presence of calcium

Protein II, a 32K cytoskeleton-associated protein isolated from porcine intestinal epithelium, binds to vesicles composed of phosphatidylserine in the presence, but not the absence, of 10 microM Ca2+. Binding was saturable and was specifically inhibited by chelation of free Ca2+ with EGTA. Binding was also inhibited by trifluophenothiazine. Vesicles composed of dimyristoylphosphatidylcholine did not bind protein II, suggesting that interaction with phosphatidylserine was selective. These properties are consistent with a possible role for protein II in Ca-regulated cytoskeleton-cell membrane events.     

Polylysine-induced 2H NMR-observable domains in phosphatidylserine/phosphatidylcholine lipid bilayers.

The interaction of three polylysines, Lys(5) (N = 5), Lys(30) (N = 30), and Lys(100) (N = 100), where N is the number of lysine residues per chain, with phosphatidylserine-containing lipid bilayer membranes was investigated using 2H NMR spectroscopy. Lys(30) and Lys(100) added to multilamellar vesicles composed of (70:30) (mol:mol) mixtures of choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) produced two resolvable 2H NMR spectral components under conditions of low ionic strength and for cases where the global anionic lipid charge was in excess over the global cationic polypeptide charge. The intensities and quadrupolar splittings of the two spectral components were consistent with the existence of polylysine-bound domains enriched in POPS, in coexistence with polylysine-free domains depleted in POPS. Lys(5), however, yielded no 2H NMR resolvable domains. Increasing ionic strength caused domains to become diffuse and eventually dissipate entirely. At physiological salt concentrations, only Lys(100) yielded 2H NMR-resolvable domains. Therefore, under physiological conditions of ionic strength, pH, and anionic lipid bilayer content, and in the absence of other, e.g., hydrophobic, contributions to the binding free energy, the minimum number of lysine residues sufficient to produce spectroscopically resolvable POPS-enriched domains on the 2H NMR millisecond timescale may be fewer than 100, but is certainly greater than 30.     

The first EGF-like domain from human factor IX contains a high-affinity calcium binding site.

It has been suggested that epidermal growth factor-like (EGF-like) domains, containing conserved carboxylate residues, are responsible for the high-affinity calcium binding exhibited by a number of vitamin K-dependent plasma proteins involved in the control of the blood coagulation cascade. These include the procoagulant factors IX and X, and the anticoagulants protein C and protein S. To test this hypothesis we have expressed the first EGF-like domain from human factor IX (residues 46-84) using a yeast secretion system, and examined calcium binding to the domain. Using 1H-NMR to measure a calcium-dependent shift assigned to Tyr69 we have detected a high-affinity calcium binding site (Kd = 200-300 microM). We suggest that other EGF-like domains of this type may have similar calcium binding properties. In addition, we have completely assigned the aromatic region of the NMR spectrum by NOESY and COSY analysis, and have used these data to discuss the effect of calcium and pH on the conformation of the domain with reference to a model based on the structure of human EGF.     

Bioconversion of phosphatidylcholine to phosphatidylserine using immobilized enzyme mini-columns

Phospholipase-d, immobilized on controlled porosity glass beads, was used for the preparation of phosphatidylserine. The synthesis of phosphatidylserine was monitored using immobilized mini-reactors of choline oxidase and serine dehydratase isolated from rat liver. The immobilized enzymes showed good stability, and no deterioration in enzyme activity was recorded after use for 4 months.     

A spin-label study of phosphatidylcholine exchange protein. Regulation of the activity by phosphatidylserine and calcium ion.

The effects of the divalent cations Mg2+, Mn2+ and Ca2+ on the Brownian rotational motion of fluorescently labeled myosin, heavy meromyosin and myosin subfragment-1 were measured by the method of time-resolved fluorescence depolarization. When Mg2+ was added to solutions of myosin or heavy meromyosin and EDTA, their rotational mobility increased. Ca2+ had no effect. Mn2+ increased the mobility of heavy meromyosin but decreased that of myosin. None of these divalent cations effected the mobility of subfragment-1. The binding of heavy meromyosin to actin was affected very little by Mg2+ or EDTA over a wide range of conditions. Divalent cations appear to change the swivel about which the heads of myosin rotate, presumably by binding to light chain 2 (also called DTNB light chain). However, the heads are still able to bind actin in nearly the same way whether Mg2+ is present or not. The concentration of free Mg2+ for the mid-point of the change in heavy meromyosin mobility is in good agreement with that for EDTA activation of ATPase activity. This suggests that EDTA activation is due to removal of Mg2+ bound to myosin itself.     

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)     

Binding of spin-labeled local anesthetics to phosphatidylcholine and phosphatidylserine liposomes

A homologous series of spin-labeled local anesthetics, 2-[N-methyl N-(2,2,6,6-tetramethylpiperidinooxyl)] ethyl-p-alkoxybenzoates were shown to bind to phosphatidylcholine and phosphatidylserine liposomes. Under similar conditions, 70% of the ethoxy homolog (R2C) of these spin-labeled local anesthetics bound to synthetic dipalmitoyl lecithin while 98% bound to phosphatidylserine liposomes. Five percent of R2C's bound signal could be released by 4 mm calcium from phosphatidylserine liposomes, but calcium had no effect on R2C bound to synthetic lecithin. The butoxy (R4C) and hexyloxy (R6C) homologs bound to phosphatidylcholine in the order R6C > R4C. All of R6C and all of R4C were bound to phosphatidylserine liposomes, while only 90% of R6C bound to synthetic dipalmitoyl lecithin. Calcium was incapable of displacing bound R4C or R6C from either phosphatidylcholine or phosphatidylserine liposomes. The results are discussed in light of anesthetic binding by electrostatic and Van der Waal's forces to phospholipids.     

Phospholipid binding of annexin V: effects of calcium and membrane phosphatidylserine content.

We studied the binding of fluorescein-labeled annexin V (placental anticoagulant protein I) to small unilamellar phospholipid vesicles at 0.15 M ionic strength as a function of calcium concentration and membrane phosphatidylserine (PS) content. As the mole percentage of PS in the membrane increased from 10 to 50%, the stoichiometry of binding decreased hyperbolically from 1100 mol phospholipid/mol annexin V to a limiting value of 84 mol/mol for measurements made at 1.2 mM CaCl2. Over the same range of PS content, Kd remained approximately constant at 0.036 +/- 0.011 nM. A similar hyperbolic decrease in stoichiometry was observed with vesicles containing 10 or 20% PS when the calcium concentration was increased from 0.4 to 10 mM. Thus, the density of membrane binding sites is strongly dependent on the membrane PS content and calcium concentration. The effect of calcium on annexin V-membrane binding is proposed to be due to the formation of phospholipid-calcium complexes, to which the protein binds, rather than to an allosteric effect of calcium on protein-phospholipid affinity.     

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.     

Volume changes in high-affinity calcium binding of the sarcoplasmic reticulum calcium-transport enzyme.

The effect which hydrostatic pressure exerts on the hydrolysis of dinitrophenyl phosphate and nitrophenyl phosphate by the sarcoplasmic reticulum calcium-transport enzyme was determined. Activation volumes for substrate hydrolysis at saturating and non-saturating concentrations of calcium were determined and used to evaluate volume increments for initial calcium binding. A reaction scheme in which two unidirectional substrate-driven reactions transfer high-affinity into low-affinity calcium-binding sites was applied to determine binding-volume increments. It has been inferred from the pressure dependence of the volume-generating function, defined as the difference between the reciprocal reaction rates of the saturated and the unsaturated enzyme, that calcium binding proceeds in two steps. The two associated binding constants are endowed with large binding-volume increments of opposite signs (+84 to +207 ml/mol and -3 to -136 ml/mol). Under different experimental conditions, with respect to the temperature, degree of calcium saturation and absence or presence of Me2SO, they add up to the same integral volume increment of 73 +/- 3.5 ml/mol for the entry of two calcium ions into the reaction cycle. In aqueous media, the two binding constants contribute about equally to binding and to the observed binding-volume increment. The presence of Me2SO strongly favours the first binding step. The size of the integral volume increment is in line with that determined for the interaction of calcium with calmodulin [Kupke, D.W. & Dorrier, T.E. (1986) Biochem. Biophys. Res. Commun. 38, 199-204].     

Fluorescence measurements of the binding of cations to high-affinity and low-affinity sites on ATP-G-actin

The binding of cations to ATP-G-actin has been assessed by measuring the kinetics of the increase in fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Ca2+ and Mg2+ compete for a single high-affinity site on ATP-G-actin with KD values of 1.5-15 nM for Ca2+ and 0.1-1 microM for Mg2+, i.e. with affinities 3-4 orders of magnitude higher than previously reported (Frieden, C., Lieberman, D., and Gilbert, H. R. (1980) J. Biol. Chem. 255, 8991-8993). As proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886), the Mg-actin complex undergoes a slow isomerization (Kis = 0.03-0.1) to a higher affinity state (K'D = 4-40 nM). The replacement of Ca2+ by Mg2+ at this high-affinity site causes a slow 10% increase in fluorescence that is 90% complete in about 200 s at saturating concentrations of Mg2+. Independently, Ca2+, Mg2+, and K+ bind to low-affinity sites (KD values of 0.15 mM for Ca2+ and Mg2+ and 10 mM for K+) which causes a rapid 6-8% increase in fluorescence (complete in less than 5 s). We propose that the activation step that converts Ca-G-actin to a polymerizable species upon addition of Mg2+ is the binding of Mg2+ to the low-affinity sites and not the replacement of Ca2+ by Mg2+ at the high-affinity site.