A domain of membrane-bound blood coagulation factor Va is located far from the phospholipid surface. A fluorescence energy transfer measurement

The larger subunit of blood coagulation factor Va was covalently labeled with iodoacetamido derivatives of fluorescein and rhodamine without loss of functional activity, as measured by either the one-stage clotting assay or the ability to accelerate prothrombin activation in a purified system. The spectral properties of the dyes were not altered by the presence or absence of the smaller subunit of factor Va, Ca2+, prothrombin, factor Xa, or phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) vesicles. When fluorescein-labeled protein (factor VaF) was titrated with PC/PS vesicles containing either octadecylrhodamine or 5-(N-hexadecanoylamino)eosin, fluorescence energy transfer was observed between the protein-bound donor dyes and the acceptor dyes at the outer surface of the phospholipid bilayer. The extent of energy transfer correlated directly with the extent of protein binding to the vesicles monitored by light scattering. The distance of closest approach between the fluorescein on factor Va and the bilayer surface averaged 90 A for the two different acceptors. Association of factor VaF with factor Xa on the phospholipid surface reduced this separation by 7 A, but association with prothrombin did not alter the distance between the labeled domain on factor VaF and the surface. The efficiency of diffusion-enhanced energy transfer between rhodamine-labeled factor Va and terbium dipicolinate entrapped inside PC/PS vesicles was less than 0.01, consistent with the location of the dye far above the inner surface of the vesicle. Thus, a domain of membrane-bound factor Va is located a minimum of 90 A above the phospholipid surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic properties of the yeast phospholipid transfer protein

The properties of the phosphatidylcholine (PC) transfer reaction catalyzed by the yeast phospholipid transfer protein (TP-I) were examined in vitro. Donor and acceptor membranes consisted of unilamellar (ULV) and multilamellar (MLV) vesicles, respectively. The phospholipid composition of the membranes participating in the transfer reaction, and in particular that of the MLV acceptors, have a tremendous effect upon the rate of PC-catalyzed transfer. Phosphatidylethanolamine (PE) is an essential component of the acceptor membrane, but it alone is not sufficient to sustain appreciable transfer rates. If combined in an equimolar ratio with PC, there is only a modest increase in transfer rates. On the other hand, when combined with alternate substrates such as phosphatidylinositol (PI) or phosphatidylserine (PS), very high rates of PC transfer occur. The measurement of transfer rates is not affected by the molecular species of PC used as the radioactive tracer. Evidence is also presented to indicate that the two forms of the transfer protein (TP-I and TP-II) are not identical in terms of their interactions with a membrane surface: differences occur in the levels of transfer of PC, PE, PI, and PS at equilibrium. Finally, by kinetic analysis, the mechanism of the protein-catalyzed transfer of PC is shown to conform to a ping-pong bibi model with excess substrate inhibition, analogous to ordinary two-substrate enzyme-catalyzed reactions. Both the rates of desorption and adsorption of the protein from the surface of the ULV are much greater than those describing the similar interactions of the protein with MLV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovalbumin-induced fusion of acidic phospholipid vesicles at low pH

The interactions of ovalbumin (OA) with large unilamellar vesicles (LUV) of phosphatidylserine (PS) and PS/phosphatidylethanolamine (PE) were studied. It was observed that OA induces aggregation, destabilization, and fusion of these LUV composed of acidic phospholipids at low pH levels. The fusion of LUV by OA was monitored by measuring the intermixing of internal aqueous contents of vesicles, by resonance energy transfer assay which follows the mixing of the membrane components, and by thin-sectioning electron microscopy. The pH profile of fusion was found to be similar to the pH-dependent binding of OA to the same phospholipid vesicles. Proteolytic digestion and hydrophobic labeling with dansyl chloride and photoreactive phosphatidylcholine (PC) of the OA-vesicle complex showed that a segment of OA with a molecular weight of approximately 2,500 penetrates the bilayer. The amino acid composition of this segment indicated that it is the 291-322 fragment and not the putative signal sequence.     

Substrate specificity of human plasma phospholipid transfer protein

The ability of human plasma phospholipid transfer protein to transfer L-alpha-[14C]dipalmitoylphosphatidylcholine (DPPC) from donor vesicles to acceptor high-density lipoproteins (HDL) was examined, using vesicles of different compositions and sizes, and native or chemically modified HDL. Phosphatidylcholine (PC) transfer was inhibited by both cholesterol and sphingomyelin incorporation into egg-PC vesicles. On a molar basis, cholesterol inhibited transfer about 5-fold more than sphingomyelin; however, the effects of both lipids on the fluidity of the vesicle membrane (measured by fluorescence polarization of diphenylhexatriene), were closely correlated with their effects on PC transfer activity. Increase in vesicle size, and decrease in bilayer curvature, also reduced transfer: the largest vesicles had no transfer activity at all. Addition of phosphatidic acid up to 17 mol% had no effect on PC transfer. HDL apolipoprotein lysyl residues were chemically modified by reductive methylation, citraconylation, or acetoacetylation. The effects of modification on the apolipoprotein structure and on the HDL particle were assessed by intrinsic fluorescence measurements, SDS-polyacrylamide gel electrophoresis patterns, and gel chromatography. Only acetoacetylation significantly affected any of these parameters. The ability of HDL to accept PC in the absence of phospholipid transfer protein decreased with an increase in apolipoprotein negative charge while, in the presence of phospholipid transfer protein, the acceptor ability of HDL increased up to 1.7-fold with an initial increase in negative charge and then decreased, ultimately to zero, upon extensive modification.     

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.     

Asymmetric fusion between phospholipid vesicles and vesicles formed from synthetic di(n-alkyl)phosphates

We have investigated the fusion behavior of a mixed vesicle system consisting of vesicles prepared from the simple synthetic surfactants di(n-dodecyl)phosphate (DDP) or di(n-tetradecyl)phosphate (DTP) and vesicles prepared from the phospholipids phosphatidylserine (PS) or dioleoylphosphatidylcholine (DOPC). Fusion between the vesicles, induced by Ca2+, was determined by a resonance energy transfer assay for lipid mixing, sucrose density gradient analysis, and electron microscopy. We demonstrate that synthetic surfactant vesicles can specifically engage in asymmetric fusion events, provided that the incubation temperature is kept below the gel-liquid crystalline phase-transition temperature (Tc) of the synthetic amphiphile (29 and 48 degrees C for DDP and DTP, respectively) and that the physical state of the target membrane is fluid. Asymmetric fusion of DDP or DTP vesicles was most efficient with PS vesicles, but it also occurred with zwitterionic PC vesicles. In the latter case, fusion proceeded spontaneously, but the process was markedly accelerated upon addition of Ca2+. Furthermore, in contrast to a massive transformation of bilayer into nonbilayer hexagonal HII tubular structures, as occurs upon symmetric Ca(2+)-induced fusion of DDP vesicles, asymmetric fusion with phospholipid bilayers predominantly leads to the formation of larger vesicles. This indicates that both PS and DOPC stabilize the DDP bilayer structure in the fusion product.     

Association of protein kinase C with phospholipid vesicles

The Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), was purified from bovine brain by a modified procedure that provided sufficient quantities of stable protein for analysis of physical properties of protein-membrane binding. The binding of PKC to phospholipid vesicles of various compositions was investigated by light-scattering and fluorescence energy transfer measurements. The binding properties for membranes of low phosphatidylserine (PS) content were consistent with a peripheral membrane association; PKC showed Ca2+ -dependent binding to phospholipid vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylglycerol. Membranes containing 0-20% PS (the remainder of the phospholipid was phosphatidylcholine) bound less protein than membranes containing greater than 20% PS; the factor limiting protein binding to membranes containing low PS appeared to be the availability of acidic phospholipids. Increasing the PS content above 20% did not increase the amount of membrane-bound protein at saturation, and the limiting factor was probably steric packing of protein on the membrane surface. The membranes bound about 1 g of protein/g of phospholipid at steric saturation. Binding was of relatively high affinity (Kd less than 5 nM), and the association rate was rapid on the time scale of the experiments. Addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to phospholipid-bound PKC caused dissociation of the complex, and the properties of this dissociation indicated an equilibrium binding of protein to membrane. However, only partial dissociation of PKC was achieved when the PS content of the vesicles exceeded 20%. A number of comparisons revealed that binding of protein to the membrane, even in the presence of phorbol esters, was insufficient for development of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification, characterization and substrate specificity of rabbit lung phospholipid transfer proteins.

Three phospholipid transfer proteins, namely proteins I, II and III, were purified from the rabbit lung cytosolic fraction. The molecular masses of phospholipid transfer proteins I, II and III are 32 kilodaltons (kDa), 22 kDa and 32 kDa, respectively; their isoelectric point values are 6.5, 7.0 and 6.8, respectively. Phospholipid transfer proteins I and III transferred phosphatidylcholine (PC) and phosphatidylinositol (PI) from donor unilamellar liposomes to acceptor multilamellar liposomes; protein II transferred PC but not PI. All the three phospholipid transfer proteins transferred phosphatidylethanolamine poorly and showed no tendency to transfer triolein. The transfer of [14C]PC from unilamellar liposomes to multilamellar liposomes facilitated by each protein was affected differently by the presence of acidic phospholipids in the PC unilamellar liposomes. In an equal molar ratio of acidic phospholipid and PC, phosphatidylglycerol (PG) reduced the activities of proteins I and III by 70% (P = 0.0004 and 0.0032, respectively) whereas PI and phosphatidylserine (PS) had an insignificant effect. In contrast, the protein II activity was stimulated 2-3-times more by either PG (P = 0.0024), PI (P = 0.0006) or PS (P = 0.0038). In addition, protein II transferred dioleoylPC (DOPC) about 2-times more effectively than dipalmitoylPC (DPPC) (P = 0.0002), whereas proteins I and III transferred DPPC 20-40% more effectively than DOPC but this was statistically insignificant. The markedly different substrate specificities of the three lung phospholipid transfer proteins suggest that these proteins may play an important role in sorting intracellular membrane phospholipids, possibly including lung surfactant phospholipids.     

Prothrombin activation on phospholipid membranes with positive electrostatic potential

The conversion of prothrombin into thrombin, which is a crucial reaction in hemostatic plug formation, is greatly stimulated by phospholipids plus calcium ions. It has been proposed that phospholipid surfaces which promote blood coagulation should have a negative surface charge [Bangham, A. D. (1961) Nature (London) 192, 1197-1198]. However, the experiments that led to this proposal were carried out with one kind of anionic phospholipid (dicetyl phosphate). Here we report that membranes, which contain phosphatidylserine (PS) as the anionic phospholipid, can be made positively charged by incorporation of stearylamine and still exhibit almost full procoagulant and prothrombin-converting activity. This suggests that electrostatic forces contribute negligibly to the binding of coagulation factors to PS-containing membranes. Introduction of stearylamine in membranes containing phosphatidyl-beta-lactate (PLac) causes considerable inhibition of their prothrombin-converting activity. Since PLac and PS only differ by the presence of an amino group in the polar head group, the much higher procoagulant activity of PS-containing vesicles is indicative of an important function of the amino group of PS in the interaction with coagulation factors. We propose that the association of coagulation factors with PS-containing membranes results from complex formation between Ca2+ ions and ligands supplied by the protein and by PS molecules. The ability to form such a complex may well explain why cell membranes with PS have such excellent procoagulant properties.     

Glucosylceramide,a neutral glycosphingolipid anticoagulant cofactor,enhances the interaction of human- and bovine-activated protein C with negatively charged phospholipid vesicles

The effect of glucosylceramide (GlcCer) on activated protein C (APC)-phospholipid interactions was examined using fluorescence resonance energy transfer. Human APC, labeled with either fluorescein (Fl-APC) or dansyl (DEGR-APC) donor, bound to phosphatidylcholine/phosphatidylserine (PC/PS, 9:1 w/w) vesicles containing octadecylrhodamine (OR) acceptor with a K(d) (app) = 16 micro g/ml, whereas Fl-APC (or DEGR-APC) bound to PC/PS/GlcCer(OR) (8:1:1) vesicles with a K(d) (app) = 3 micro g/ml. This 5-fold increase in apparent affinity was not species-specific since bovine DEGR-APC also showed a similar GlcCer-dependent enhancement of binding of APC to PC/PS vesicles. From the efficiency of fluorescence resonance energy transfer, distances of closest approach of approximately 63 and approximately 64 A were estimated between the dansyl on DEGR-APC and rhodamine in PC/PS/GlcCer(OR) and PC/PS(OR), respectively, assuming kappa(2) = 2/3. DEGR-APC bound to short chain C8-GlcCer with an apparent K(d) of 460 nm. The presence of C8-GlcCer selectively enhanced the binding of C16,6-NBD-phosphatidylserine but not C16,6-7-nitrobenz-2-oxa-1,3-diazole (NBD)-phosphatidylcholine to coumarin-labeled APC. These data suggest that APC binds to GlcCer, that PC/PS/GlcCer vesicles like PC/PS vesicles bind to the N-terminal gamma-carboxyglutamic acid domain of APC, and that one mechanism by which GlcCer enhances the activity of APC is by increasing its affinity for membrane surfaces containing negatively charged phospholipids.     

The cytoplasmic region of mouse Fc gamma RIIb1, but not Fc gamma RIIb2, binds phospholipid membranes

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, Fc gamma RIIb1 and Fc gamma RIIb2, play key roles in signal transduction by mediating different cellular functions. The Fc gamma RIIb1 (94 residues) and Fc gamma RIIb2 (47 residues) cytoplasmic regions are generated by differential mRNA splicing in which a single aspartic acid residue in Fc gamma RIIb2 is replaced by a 48-residue insert in Fc gamma RIIb1. In previous work, quantities of the mFc gamma RIIb1 and mFc gamma RIIb2 cytoplasmic regions were generated, and their secondary structures were examined in different solutions with circular dichroism [Chen, L., Thompson, N. L., and Pielak, G. J. (1997) Protein Sci. 6, 1038-1046]. In the work described here, steady-state light scattering was used to investigate possible interactions of the two isolated cytoplasmic regions with phospholipid vesicles. Three phospholipid compositions were examined: phosphatidylserine/phosphatidylcholine (PS/PC) (25/75, mol/mol); phosphatidylinositol bisphosphate/phosphatidylcholine (PIP2/PC) (25/75, mol/mol); and pure phosphatidylcholine (PC). Binding was examined in the presence and absence of Ca2+. The mFc gamma RIIb1 cytoplasmic peptide binds PS/PC vesicles weakly in the absence of Ca2+ and more strongly in the presence of Ca2+. For PIP2/PC vesicles, the behavior is reversed; binding is weak in the presence of Ca2+ and stronger in its absence. The mFc gamma RIIb1 peptide also weakly binds pure PC vesicles, in a Ca2+-independent manner. The mFc gamma RIIb2 cytoplasmic peptide does not bind, in the presence or absence of Ca2+, to PS/PC, PIP2/PC, or PC vesicles. The implications of these results for the mechanisms of signal transduction mediated by the two mFc gamma RII cytoplasmic regions are discussed.     

Site-site interaction in the phospholipid activation of D-beta-hydroxybutyrate dehydrogenase

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with absolute specificity for phosphatidylcholine (PC). The enzyme devoid of lipid, the apodehydrogenase, inserts spontaneously into phospholipid vesicles where it exists as a tetramer. We now find the lipid activation to be limited by the mole fraction of PC in the total phospholipid. These studies suggest that the concentration of the enzyme-PC complex, which is essential for enzymic activity, becomes diffusion limited at lower PC concentration. The lipid activation and the tryptophan fluorescence of purified D-beta-hydroxybutyrate dehydrogenase were studied in the presence of a constant "bilayer background" of approximately 100 nonactivating phospholipid molecules/enzyme monomer. Activation by PC was half-maximal at 20 PC molecules/enzyme monomer. This value was doubled when the amount of "background" phospholipid was doubled. Activation proceeded with positive cooperativity having a Hill coefficient of approximately 2.4. These data indicate interactions between at least three PC-binding sites. The quenching of tryptophan fluorescence by the phospholipid activator, 1-palmitoyl-2-(1-pyrenyl)-decanoyl-PC (2-pyrenyl-PC), gives a saturation curve with half-maximal quenching of 6 quencher molecules/enzyme monomer. This value is equivalent to an apparent phospholipid-protein dissociation constant in the two-dimensional membrane and corresponds to approximately 6 mol % of total phospholipid. In distinct contrast to the phospholipid activation curve, the fluorescence quenching saturation curve was hyperbolic and there was no specificity for PC. The fluorescence quenching by 2-pyrenyl-PC could be diminished by using a several-fold excess of PC or other phospholipids so as to reduce the mole fraction of quencher in the bilayer. It would appear that formation of enzyme-PC complex is a dynamic process consisting of at least two discernible steps: 1) a primary interaction, as measured by tryptophan quenching, which is hyperbolic and not specific for lecithin. This interaction is independent from and precedes 2) phospholipid activation of D-beta-hydroxybutyrate dehydrogenase, which is cooperative in nature and specific for lecithin.     

Influence of membrane phospholipid composition and structural organization on spontaneous lipid transfer between membranes

Investigations were carried out on the influence of phospholipid composition of model membranes on the processes of spontaneous lipid transfer between membranes. Acceptor vesicles were prepared from phospholipids extracted from plasma membranes of control and ras-transformed fibroblasts. Acceptor model membranes with manipulated levels of phosphatidylethanolamine (PE), sphingomyelin and phosphatidic acid were also used in the studies. Donor vesicles were prepared of phosphatidylcholine (PC) and contained two fluorescent lipid analogues, NBD-PC and N-Rh-PE, at a self-quenching concentration. Lipid transfer rate was assessed by measuring the increase of fluorescence in acceptor membranes due to transfer of fluorescent lipid analogues from quenched donor to unquenched acceptor vesicles. The results showed that spontaneous NBD-PC transfer increased upon fluidization of acceptor vesicles. In addition, elevation of PE concentration in model membranes was also accompanied by an increase of lipid transfer to all series of acceptor vesicles. The results are discussed with respect to the role of lipid composition and structural order of cellular plasma membranes in the processes of spontaneous lipid exchange between membrane bilayers.     

Kinetics of phospholipid exchange between bilayer membranes.

In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469,311--325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, k--, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are kP- = (0.86 +/- 0.05) - 10(-5) S-1 and ke- = (1.09 +/- 0.13) - 10(-6) s-1 for phospholipid molecules with trans-delta 9-hexadecenoate and trans-delta 9-octadecenoate, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids.     

The physical significance of Km in the prothrombinase reaction

Key kinetic parameters for the prothrombinase complex formed on membranes of phosphatidylserine (PS)/phosphatidylcholine (PC) (40/60) (Km = 0.12 microM, kcat = 11 s-1) or PS/PC (2/98) (Km = 0.40 microM, kcat = 11 s-1) differed only slightly. In contrast, the density of proteins on the membrane surface at the km differed greatly for the two membranes. The kinetics appeared unaffected by conditions where the number of phospholipid vesicles (2% PS) exceeded the number of protein molecules. These results establish that the Km for the prothrombinase reaction is determined by the concentration of prothrombin in solution rather than its density at the membrane surface. This system can be treated as a dissociable enzyme acting on a soluble substrate.     

Human factor VIII procoagulant activity and phospholipid interaction

The interaction between purified human factor VIII and phospholipid vesicles was investigated. The binding of factor VIII to an equimolecular mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) was studied by sucrose gradient ultracentrifugation (10–40% w/v saccharose in 0.01 M Tris-HCl/0.15 M NaCl buffer (pH 7). In the absence of phospholipids all factor VIII activities (VIII : C, VIII R : WF and VIII R : AG) were found in the zone of highest sucrose density including the factor VIII related protein subunit (200 000 molecular weight). In the presence of an equimolecular mixture of PS/PC VIII R : WF activity, VIII R : AG and a factor VIII related protein still migrated to the bottom of the tube, while VIII : C activity remained at the top where phospholipids were found. Thus a dissociation phenomenon between VIII : C and the other factor VIII relateda activities was apparent in the presence of phospholipids. These results also demonstrate the binding of factor VIII : C to certain active phospholipids.     

Association of lysozyme with phospholipid vesicles is accompanied by membrane surface dehydration

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between binding of the protein and the subsequent destabilization of the phospholipid vesicles a set of experiments was performed using phospholipid monolayers and vesicles. Using microelectrophoresis the binding of lysozyme to phospholipid vesicles made of PS was determined. At low ionic strength and mild acidic pH of the solution lysozyme reduced the magnitude of the negative zeta potential of PS vesicles at lower concentrations compared to neutral pH and high ionic strength. In contrast, the bound fraction of lysozyme to PS vesicles was nearly constant at acidic and neutral pH. At low pH, the binding of lysozyme was accompanied by a strong aggregation of the vesicles. Lysozyme binding to PS vesicles is accompanied by its penetration into the PL monolayer. This was measured by surface tension and film balance measurements at low pH and low ionic strength. The interaction of lysozyme with negatively charged vesicles lead to a decrease of the vesicle surface hydration as measured by the shift of the emission peak of the fluorescent probe DPE. The binding of bis-ANS increased at low pH after addition of lysozyme to the vesicles. This indicates that more hydrophobic patches of the lysozyme-PS complex are exposed at low pH. At low pH the binding process of lysozyme to PS vesicles was followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the aqueous content of vesicles.     

Effect of acceptor membrane phosphatidylcholine on the catalytic activity of bovine liver phosphatidylcholine transfer protein

Protein-mediated transfer of phosphatidylcholine (PC) by bovine liver phosphatidylcholine transfer protein (PC-TP) was examined using a vesicle-vesicle assay system. Donor and acceptor membranes were prepared from Escherichia coli phospholipids and limiting amounts of egg yolk PC. PC transfer between vesicles of E. coli lipid/egg PC was markedly higher than transfer of PC from vesicles of E. coli lipid/egg PC to vesicles of E. coli lipid. Kinetic parameters of the interaction between PC-TP and E. coli lipid vesicles with or without PC was investigated. The apparent dissociation constants of the complex formed between PC-TP and these vesicles were determined kinetically and from double-reciprocal plots of intrinsic PC-TP fluorescence intensity increase versus vesicle concentration. The magnitude of the dissociation constant decreased as the PC content of the vesicles increased from 0 to 5 mol%. In addition, kinetic analysis revealed that the presence of PC in acceptor vesicles increased both the association and dissociation of PC-TP from vesicles. The effect of membrane PC molecules on transfer rates was examined using bis-phosphatidylcholine, a dimeric PC molecule which is not transferred by PC-TP. Rates of PC transfer to acceptor vesicles comprised of E. coli lipid/bis-PC were virtually identical to rates observed with acceptors vesicles prepared from E. coli lipid. The results suggest that transfer of PC by PC-TP is enhanced only when insertion of protein-bound PC occurs concurrently with the extraction of a molecule of membrane PC, i.e., a concerted, one-step catalytic mechanism for phospholipid exchange.     

Effects of apolipoprotein structure on the kinetics of apolipoprotein transfer between phospholipid vesicles

The kinetics and mechanism of transfer of 14C-labeled human apolipoproteins A-I, A-II and C-III1 between small unilamellar vesicles (SUV) have been investigated. Ion exchange chromatography was used for rapid separation of negatively charged egg phosphatidylcholine (PC)/dicetyl phosphate donor SUV containing bound 14C-labeled apoprotein from neutral egg PC acceptor SUV present in 10-fold molar excess. The transfer kinetics of these apolipoproteins at 37 degrees C are consistent with the existence of fast, slow and apparently 'nontransferrable' pools of SUV-associated lipoprotein: the transfers from these pools occur on timescales of seconds (or less), minutes/hours and days/weeks, respectively. For donor SUV containing about 15 apoprotein molecules per vesicle and at a donor SUV concentration of 0.15 mg phospholipid/ml incubation mixture, the sizes of the fast kinetic pools for apolipoproteins A-I, A-II and C-III1 associated with donor SUV are 2, 10 and 11%, respectively. The sizes of the slow kinetic pools for these apolipoproteins are 16, 71 and 50%, respectively. The transfer of the various apolipoproteins from the slow kinetic pool follows first order kinetics and the half-time (t1/2) values are in the order: apo C-III1 less than apo A-I. Increasing the number of apoprotein molecules per donor SUV enlarges the size of the fast pool and increases the t1/2 of slow transfer. The differences in the kinetics of apolipoprotein transfer between SUV are consequences of the variations in the primary and secondary structures of the apolipoprotein molecules. The slow transfer of apoprotein molecules is mediated by collisions between donor and acceptor SUV; the rate is dependent on the apoprotein molecular weight with larger molecules transferring more slowly from donor SUV containing the same lipid/protein molar ratio. The hydrophobicity of the apoprotein molecule is also significant with less hydrophobic molecules transferring more rapidly. Further understanding of the differences in the kinetics of transfer of these apolipoproteins will require more knowledge of their secondary and tertiary structures.     

gamma-cyclodextrins greatly enhance translocation of hydrophobic fluorescent phospholipids from vesicles to cells in culture. Importance of molecular hydrophobicity in phospholipid trafficking studies

Short-chain, fluorescent derivatives are commonly used to investigate intracellular phospholipid trafficking. However, their use can yield misleading results because they, unlike the native species, can rapidly distribute between organelles due to their low hydrophobicity. On the other hand, hydrophobic derivatives are very difficult to introduce to cells and thus have hardly been used. Here we show that carboxyethylated gamma-cyclodextrin (CE-gamma-CD) greatly enhances transfer of a variety of hydrophobic fluorescent phospholipid derivatives from vesicles to cultured cells. Several lines of evidence indicate that CE-gamma-CD enhances transfer of lipid molecules by increasing their effective concentration in the aqueous phase, rather than by inducing membrane fusion or hemifusion. Incubation with CE-gamma-CD and donor lipid vesicles does not extract cholesterol or phospholipids from the cells or compromise plasma membrane intactness or long term cell viability. Using CE-gamma-CD-mediated transfer, we introduced hydrophobic pyrene-labeled phosphatidylserine to the plasma membrane of fibroblast cells and followed their distribution with time. In contrast to what has been previously observed for other, less hydrophobic species, transport of this lipid to the Golgi apparatus or mitochondria was not detected. Rather, much of this fluorescent PS remained in the plasma membrane or was incorporated to various endocytotic compartments. These findings indicate that the native, typically hydrophobic phosphatidylserine molecules efflux only very slowly via the cytoplasm to intracellular organelles. This helps to explain how cells can maintain a very high concentration of phosphatidylserine in the inner leaflet of their plasma membrane. Furthermore, the present results underline the importance of using hydrophobic analogues when studying intracellular trafficking of many phospholipid classes.