Phospholipid transfer between phosphatidylcholine-taurocholate mixed micelles

The transfer of fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE) between phosphatidylcholine-taurocholate mixed micelles was measured by monitoring the increase in fluorescence as N-NBD-PE, initially contained in mixed micelles at self-quenching concentrations, was diluted into unlabeled mixed micelles. The half-times for transfer of a homologous series of N-NBD-PEs differing in saturated acyl chain length from 11 to 16 carbons increased with acyl chain length from 4 to 35 s. The half-times for transfer of the same N-NBD-PEs between phosphatidylcholine vesicles without taurocholate were 200-6000 times slower than those between the mixed micelles. A kinetic analysis of initial transfer rate data was used to determine the mechanistic model that best described the data. According to this analysis, the increased rate of intermicellar phospholipid transfer relative to that of intervesicular transfer is a result of (1) exchange between micelles during transient micelle collisions which is not observed between vesicles and (2) an increased rate of monomer diffusion due to a faster rate of phospholipid dissociation from mixed micelles into the water phase than from vesicles. The relative significance of dissociation from mixed micelles into the water phase than from vesicles. The relative significance of collision-dependent versus monomer diffusion transfer increases with acyl chain length and hydrophobicity.     

Lymphoma-vesicle interactions: vesicle adsorption, membrane fragmentation, and intermembrane protein transfer

Sonicated dimyristoylphosphatidylcholine vesicles interact with cultured murine lymphoma (BL/VL3) to generate complexes of vesicle and cell membrane components. Cell-free supernatants harvested after cell-vesicle incubations contain three distinct lipid species that can be separated by density gradient centrifugation. Analysis of protein and lipid composition and assays for cell and vesicle lumen contents reveal that the densest of the three lipid species comprises sealed plasma membrane fragments complexed with vesicles, while the least dense species is indistinguishable from pure phospholipid vesicles. The third, intermediate density species consists of topologically intact vesicles with associated plasma membrane proteins but without detectable cell lipids or cytoplasmic components. The membrane fragmentation and cell-to-vesicle protein transfer observed during lymphoma-vesicle incubations are examined as functions of cell and vesicle concentrations and incubation time.     

II. Phospholipid exchange and size enlargement in sonicated vesicles induced by a ‘critical’ fatty acid concentration

Size enlargement of dipalmitoyl phosphatidylcholine vesicles was greatly accelerated in the range of the phase-transition temperatures, when fatty acid concentration was above a threshold level (‘critical’ concentration). This ‘critical’ concentration varied with the length of the fatty acid chain. The size enlargement process had second-order kinetics dependent on the vesicle concentration. Alkaline pH and low ionic strength inhibited the rate of size enlargement.Phospholipid exchange between dimyristoyl and dipalmitoyl phosphatidylcholine vesicles increased abruptly above a ‘critical’ fatty acid concentration. The donor vesicles were those vesicles in which fatty acids reached the ‘critical’ concentration. The phospholipid exchange occurred both in fluid- and in solid-state vesicles. The ‘critical’ fatty acid concentration accelerating the phospholipid exchange process was lower than that accelerating the size enlargement process.The phospholipid exchange process explained in terms of a diminished hydrophobic attraction among the phospholipid molecules of the bilayer occurs via a free phospholipid molecule transfer through the aqueous phase. The size enlargement process is interpreted in terms of high fatty acid concentration in the membrane fluid domains. The membrane structure is locally perturbed inducing vesicle sticking after collision.     

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.     

Mechanism of spontaneous, concentration-dependent phospholipid transfer between bilayers

We have previously demonstrated that spontaneous phospholipid transfer between bilayer vesicles at higher vesicle concentrations is characterized not only by a first-order desorption rate but also by a second-order process dependent on vesicle concentration (Jones & Thompson, 1989b). We have extended our studies to examine the mechanism of this second-order process by investigating transfer as a function of lipid type, temperature, aqueous medium composition, and vesicle size. The results suggest a mechanism of concentration-dependent transfer in which the rate of lipid monomer desorption from vesicle bilayers is enhanced in transient vesicle-vesicle complexes.     

Interaction of intestinal brush border membrane vesicles with small unilamellar phospholipid vesicles. Exchange of lipids between membranes is mediated by collisional contact

The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second-order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision-induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions.     

Thermodynamics and kinetics of phospholipid monomer-vesicle interaction

Resonance energy transfer between acyl chain labeled (7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylcholine (NBD-PC) and head group labeled (lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE) was used to monitor the rate of NBD-PC transfer between two populations of dioleoylphosphatidylcholine (DOPC) vesicles. Equilibration of NBD-PC between DOPC vesicles occurs by the diffusion of soluble monomers through the water phase, which is a first-order process. Conditions were used such that the apparent transfer rate constant is equal to the rate constant for monomer-vesicle dissociation into solution. The partition distribution of NBD-PC between DOPC vesicles and water was determined by measuring the loss of NBD-PC from vesicles into solution following the dilution of small amounts of vesicles in buffer. The acyl chain length and temperature dependence of both the rate and partition measurements were determined, and a free energy diagram for NBD-PC-soluble monomer-vesicle interactions was constructed. The conclusions of this analysis are the following: NBD-PC dissociation from and association with the bilayer require passage through a high-energy transition state resulting predominantly from enthalpic energy. The activation energy for NBD-PC-vesicle dissociation becomes more positive and the standard free energy of NBD-PC transfer from water to vesicles becomes more negative with increasing acyl chain length. The standard free energy of transfer for NBD-PC from water to vesicles results predominantly from differences in enthalpy between the membrane and water phases. The enthalpy of activation for association increases with acyl chain length and is larger than expected for an aqueous diffusion-limited process in bulk water.     

Mechanism of the cell-penetrating peptide transportan 10 permeation of lipid bilayers

The mechanism of the interaction between the cell-penetrating peptide transportan 10 (tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of peptide association with vesicles and peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence. The experimental kinetics were analyzed by directly fitting to the data the numerical solution of mathematical kinetic models. A very good global fit was obtained using a model in which tp10 binds to the membrane surface and perturbs it because of the mass imbalance thus created across the bilayer. The perturbed bilayer state allows peptide monomers to insert transiently into its hydrophobic core and cross the membrane, until the peptide mass imbalance is dissipated. In that transient state tp10 "catalyzes" dye efflux from the vesicle lumen. These conclusions are consistent with recent reports that used molecular dynamics simulations to study the interactions between peptide antimicrobials and phospholipid bilayers. A thermodynamic analysis of tp10 binding and insertion in the bilayer using water-membrane transfer hydrophobicity scales is entirely consistent with the model proposed. A small bilayer perturbation is both necessary and sufficient to achieve very good agreement with the model, indicating that the role of the lipids must be included to understand the mechanism of cell-penetrating and antimicrobial peptides.     

Kinetics of fluorescent-labeled phosphatidylcholine transfer between nonspecific lipid transfer protein and phospholipid vesicles

Recently, rat liver nonspecific lipid transfer protein (nsLTP) was shown to form a fluorescent complex when allowed to equilibrate with self-quenching vesicles prepared from the fluorescent phospholipid 1-palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4- yl)amino]dodecanoyl]phosphatidylcholine (P-C12-NBD-PC) [Nichols, J. W. (1987) J. Biol. Chem. 262, 14172-14177]. Investigation of the mechanism of complex formation was continued by studying the kinetics of transfer of P-C12-NBD-PC between nsLTP and phospholipid vesicles using a transfer assay based on resonance energy transfer between P-C12-NBD-PC and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine. The principles of mass action kinetics (which predict initial lipid transfer rates as a function of protein and vesicle concentration) were used to derive equations for two distinct mechanisms: lipid transfer by the diffusion of monomers through the aqueous phase and lipid transfer during nsLTP-membrane collisions. The results of these kinetics studies indicated that the model for neither mechanism alone adequately predicted the initial rates of formation and dissolution of the P-C12-NBD-PC-nsLTP complex. The initial rate kinetics for both processes were predicted best by a model in which monomer diffusion and collision-dependent transfer occur simultaneously. These data support the hypothesis that the phospholipid-nsLTP complex functions as an intermediate in the transfer of phospholipids between membranes.     

Intermembrane protein transfer. Band 3, the erythrocyte anion transporter, transfers in native orientation from human red blood cells into the bilayer of phospholipid vesicles

Band 3, the erythrocyte anion transporter, has been shown to transfer between human erythrocytes and sonicated vesicles (Newton, A. C., Cook, S. L., and Huestis, W. H. (1983) Biochemistry 22, 6110-6117). Functional band 3 becomes associated with dimyristoylphosphatidylcholine vesicles incubated with human red blood cells. Proteolytic degradation patterns reveal that the transporter is transferred to the vesicles in native orientation. In erythrocytes, native band 3 is degraded on the exoplasmic membrane face by chymotrypsin and on the cytoplasmic surface by trypsin (Cabantchik, Z. I., and Rothstein, A. (1974) J. Membr. Biol. 15, 227-248; Jennings, M. L., Anderson, M. P., and Monaghan, R. (1986) J. Biol. Chem. 261, 9002-9010). Band 3 in intact protein-vesicle complexes is degraded by exogenous chymotrypsin but not by trypsin. In contrast, trypsin entrapped in the lumen of the vesicles proteolyses the vesicle-bound band 3 quantitatively. Band 3 remaining in the membranes of vesicle-treated cells and in cell fragments is not degraded detectably by vesicle-entrapped trypsin. These observations indicate that band 3 is unlikely to transfer between cell and vesicle membranes via a water-soluble form or to adhere nonspecifically to the vesicle surface; the aqueous contents of vesicles and cells (or membrane fragments) are not pooled during cell-vesicle incubations, hence no cell-vesicle fusion occurs; and the band 3 associated with the sonicated vesicle fraction is inserted in the vesicle bilayer in native orientation, with its cytoplasmic segment contacting the aqueous contents of the vesicle lumen.     

Bilayer undulation dynamics in unilamellar phospholipid vesicles: Effect of temperature,cholesterol and trehalose

We report a combined dynamic light scattering (DLS) and neutron spin-echo (NSE) study on the local bilayer undulation dynamics of phospholipid vesicles composed of 1,2-dimyristoyl-glycero-3-phosphatidylcholine (DMPC) under the influence of temperature and the additives cholesterol and trehalose. The additives affect vesicle size and self-diffusion. Mechanical properties of the membrane and corresponding bilayer undulations are tuned by changing lipid headgroup or acyl chain properties through temperature or composition. On the local length scale, changes at the lipid headgroup influence the bilayer bending rigidity κ less than changes at the lipid acyl chain: We observe a bilayer softening around the main phase transition temperature T_m of the single lipid system, and stiffening when more cholesterol is added, in concordance with literature. Surprisingly, no effect on the mechanical properties of the vesicles is observed upon the addition of trehalose.     

Head group-independent interaction of phospholipids with bile salts. A fluorescence and EPR study

Bile salts are essential for phospholipid secretion into the bile. To study the relevance of the structure of phospholipids for their interaction with bile salts, we used spin-labeled or fluorescent phospholipid analogues of different head groups and acyl chain length. Those analogues form micelles in aqueous suspension. Their solubilization by bile salts resulting in the formation of mixed micelles was followed by the decrease of spin-spin interaction of spin-labeled analogues or by the relief of fluorescence self-quenching of (7-nitro-2-1,3-benzooxadiazol (NBD))-labeled analogues. Solubilization of analogue micelles occurred at and above the critical micellar concentration (CMC) of the bile salts. As revealed by stopped-flow technique, solubilization of NBD-analogues was very rapid with half times as low as 0.1 sec above the CMC of taurocholate. Both kinetics and extent of solubilization were independent of the phospholipid head group, but were significantly affected by the fatty acid chain length. Furthermore, using vesicles with varying phospholipid composition and different types of analogues in self-quenching concentrations, we could show that bile salt-mediated vesicle solubilization depended on the fatty acid chain length of phospholipids. In contrast, neither for phospholipids nor for analogues could an influence of the lipid head group on the solubilization process be observed. These findings support a head group-independent mechanism of bile salt-mediated enrichment of specific phospholipids in the bile fluid.     

Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length

The kinetics and thermodynamics of the transmembrane movement (flip-flop) of fluorescent analogs of phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were investigated to determine the contributions of headgroup composition and acyl chain length to phospholipid flip-flop. The phospholipid derivatives containing n-octanoic, n-decanoic or n-dodecanoic acid in the sn-1 position and 9-(1-pyrenyl)nonanoic acid in the sn-2 position were incorporated at 3 mol% into sonicated single-bilayer vesicles of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC). The kinetics of diffusion of the pyrene-labeled phospholipids from the outer and inner monolayers of the host vesicles to a large pool of POPC acceptor vesicles were monitored by the time-dependent decrease of pyrene excimer fluorescence. The observed kinetics of transfer were biexponential, with a fast component due to the spontaneous transfer of pyrenyl phospholipids in the outer monolayer of labeled vesicles and a slower component due to diffusion of pyrenyl phospholipid from the inner monolayer of the same vesicles. Intervesicular transfer rates decreased approx. 8-fold for every two carbons added to the first acyl chain. Correspondingly, the free energy of activation for transfer increased approx. 1.3 kcal/mol. With the exception of PE, the intervesicular transfer rates for the different headgroups within a homologous series were nearly the same, with the PC derivative being the fastest. Transfer rates for the PE derivatives were 5-to 7-fold slower than the rates observed for PC. Phospholipid flip-flop, in contrast, was strongly dependent on headgroup composition with a smaller dependence on acyl chain length. At pH 7.4, flip-flop rates increased in the order PC less than PG less than PA less than PE, where the rates for PE were at least 10-times greater than those of the homologous PC derivative. Activation energies for flip-flop were large, and ranged from 38 kcal/mol for the longest acyl chain derivative of PC to 25 kcal/mol for the PE derivatives. Titration of the PA headgroup at pH 4.0 produced an approx. 500-fold increase in the flip-flop rate of PA, while the activation energy decreased 10 kcal/mol. Increasing acyl chain length reduced phospholipid flip-flop rates, with the greatest change observed for the PC analogs, which exhibited an approx. 2-fold decrease in flip-flop rate for every two methylene carbons added to the acyl chain at the sn-1 position.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phospholipid exchange between bilayer membranes.

The mode of interaction of aqueous dispersions of phospholipid vesicles is investigated. The vesicles (average diameter 950 A) are prepared from total lipid extracts of Escherichia coli composed of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. One type of vesicle contains trans-delta 9-octadecenoate, the other type trans-delta 9-hexadecenoate as predominant acyl chain component. The vesicles show order in equilibrium disorder transitions at transition temperatures, Tt = 42 degrees C and Tt = 29 degrees C, respectively. A mixture of these vesicles is incubated at 45 degrees C and lipid transfer is studied as a function of time using the phase transition as an indicator. The system reveals the following properties: Lipids are transferred between the two vesicle types giving rise to a vesicle population where both lipid components are homogeneously mixed. Lipid transfer is asymmetric, i.e. trans-delta 9-hexadecenoate-containing lipid molecules appear more rapidly in the trans-delta 9-octadecenoate-containing vesicles than vice versa. At a given molar ratio of the two types of vesicles the rate of lipid transfer is independent of the total vesicle concentration. It is concluded that lipid exchange through the water phase by way of single molecules or micelles is the mode of communication of these negatively charged lipid vesicles.     

Partitioning of exchangeable fluorescent phospholipids and sphingolipids between different lipid bilayer environments

Exchangeable phospho- and sphingolipid probes (phosphatidylcholine, -ethanolamine, -serine, and -glycerol, phosphatidic acid, sphingomyelin, cerebroside, and sulfatide) have been synthesized in which one acyl chain is substituted with a fluorescent bimanyl, 7-(dimethylamino)coumarin-3-yl, or diphenyl-hexatrienyl group. The distribution of these probes between two different populations of lipid vesicles can be readily monitored by fluorescence intensity measurements, as described by Nichols and Pagano [Nichols, J. W., & Pagano, R. E. (1982) Biochemistry 21, 1720-1726], when one of the vesicle populations contains a low mole fraction of a nonexchangeable quencher, (12-DABS)-18-PC. The probes examined in this study exchange between phospholipid vesicles on a time scale of minutes, with kinetics indicating that the transfer process takes place by diffusion of probe monomers through the aqueous phase. As expected, lipid probes with different charges differ markedly in their equilibrium distributions between neutral and charged lipid vesicles. However, probes with different polar headgroups differ only modestly in their relative affinities for vesicles composed of "hydrogen-bonding" lipids (PE and PS) vs "non-hydrogen-bonding" lipids (PC and PG or O-methyl-PA). Probes with different headgroups also show modest, albeit reproducible, differences in their relative affinities for cholesterol-containing vs cholesterol-free PC/PG vesicles. Our results suggest that lipids with different headgroup structures may mix more nearly ideally in liquid-crystalline lipid bilayers than would be predicted from previous analyses of the phase diagrams for binary lipid mixtures.     

Fatty acid metabolism in sn-glycerol-3-phosphate acyltransferase (plsB) mutants.

Fatty acid metabolism was examined in Escherichia coli plsB mutants that were conditionally defective in sn-glycerol-3-phosphate acyltransferase activity. The fatty acids synthesized when acyl transfer to glycerol-3-phosphate was inhibited were preferentially transferred to phosphatidylglycerol. A comparison of the ratio of phospholipid species labeled with 32Pi and [3H]acetate in the presence and absence of glycerol-3-phosphate indicated that [3H]acetate incorporation into phosphatidylglycerol was due to fatty acid turnover. A significant contraction of the acetyl coenzyme A pool after glycerol-3-phosphate starvation of the plsB mutant precluded the quantitative assessment of the rate of phosphatidylglycerol fatty acid labeling. Fatty acid chain length in membrane phospholipids increased as the concentration of the glycerol-3-phosphate growth supplement decreased, and after the abrupt cessation of phospholipid biosynthesis abnormally long chain fatty acids were excreted into the growth medium. These data suggest that the acyl moieties of phosphatidylglycerol are metabolically active, and that competition between fatty acid elongation and acyl transfer is an important determinant of the acyl chain length in membrane phospholipids.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing iso-branched fatty acids. 1. Differential scanning calorimetric studies

The thermotropic phase behavior of aqueous dispersions of phosphatidylcholines containing one of a series of methyl iso-branched fatty acyl chains was studied by differential scanning calorimetry. These compounds exhibit a complex phase behavior on heating which includes two endothermic events, a gel/gel transition, involving a molecular packing rearrangement between two gel-state forms, and a gel/liquid-crystalline phase transition, involving the melting of the hydrocarbon chains. The gel to liquid-crystalline transition is a relatively fast, highly cooperative process which exhibits a lower transition temperature and enthalpy than do the chain-melting transitions of saturated straight-chain phosphatidylcholines of similar acyl chain length. In addition, the gel to liquid-crystalline phase transition temperature is relatively insensitive to the composition of the aqueous phase. In contrast, the gel/gel transition is a slow process of lower cooperativity than the gel/liquid-crystalline phase transition and is sensitive to the composition of the bulk aqueous phase. The gel/gel transitions of the methyl iso-branched phosphatidylcholines have very different thermodynamic properties and depend in a different way on hydrocarbon chain length than do either the "subtransitions" or the "pretransitions" observed with linear saturated phosphatidylcholines. The gel/gel and gel/liquid-crystalline transitions are apparently concomitant for the shorter chain iso-branched phosphatidylcholines but diverge on the temperature scale with increasing chain length, with a pronounced odd/even alternation of the characteristic temperatures of the gel/gel transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of lysophospholipid/taurodeoxycholate submicellar aggregates with phospholipid bilayers.

The equilibrium partitioning and the rate of transfer of monoacylphosphatidylethanolamines (lysoPEs) between phospholipid bilayers and lysoPE/taurodeoxycholate submicellar aggregates (SMAs) were examined with a series of environment-sensitive fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-1-monoacylphosphatidyletha nolamine (N-NBD-lysoPE) probes of differing acyl chain length. Our previous work has demonstrated the formation of SMAs between bile salts and lysophospholipids [Shoemaker & Nichols (1990) Biochemistry 29, 5837-5842]. The experiments in the current work demonstrate that SMAs can coexist with phospholipid vesicles and can function as shuttle carriers for the transfer of lysophospholipids between membranes. The formation of submicellar aggregates of N-NBD-lysoPE and taurodeoxycholate (TDC) in equilibrium with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles was determined from the increase in fluorescence generated upon addition of TDC to POPC vesicles containing 3 mol% N-NBD-lysoPE and 3 mol% N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE) as a nonextractable fluorescence energy-transfer quencher. The fraction of lysolipid extracted increased as a function of decreasing acyl chain length of the N-NBD-lysoPE molecule. The half-time for equilibration was independent of acyl chain length and averaged 44 ms at 10 degrees C. The delivery of N-NBD-lysoPE from preformed N-NBD-lysoPE/TDC SMAs into POPC vesicles containing the energy-transfer quencher N-Rh-PE was measured by the rate of fluorescence decline. The initial rate of insertion increased with decreasing acyl chain length of the N-NBD-lysoPE molecule and as a function of vesicle concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of lipids on the enzymatic activity of 6-phosphofructo-1-kinase from B. stearothermophilus

6-Phosphofructo-1-kinase (PFK-1), a major regulatory enzyme in the glycolysis pathway, is a cytoplasmic enzyme with complicated allosteric kinetics. Here we investigate the effects of lipids on the activity of PFK from Bacillus stearothermophilus (BsPFK), to determine whether BsPFK shares any of the membrane binding or lipid binding properties reported for some mammalian PFKs. Our results show that large unilamellar vesicles (LUVs) composed of either the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or of 1:1 (mole ratio) DOPC and the fatty acid, oleic acid (OA), cause a three-fold increase in V_max, depending on the lipid concentration and vesicle composition, but no change in K_m. Further studies show lipids do not reverse the allosteric inhibitory effects of phosphoenolpyruvate (PEP) on BsPFK. SDS/PAGE studies do not show significant binding of the BsPFK tetramer to the surface of the phospholipid vesicles, suggesting that modulation of catalytic activity is due to binding of lipid monomers. By simulating the kinetics of BsPFK interaction with vesicles and lipid monomers we conclude that the change in BsPFK catalytic activity with respect to lipid concentration is consistent with monomer abstraction from vesicles rather than direct uptake of lipid monomers from solution.     

Interaction of Salmonella typhimurium with phospholipid vesicles. Incorporation of exogenous lipids into intact cells.

Incubation of intact cells of Salmonella typhimurium with bilayer phospholipid vesicles results in significant transfer of vesicle lipids to the cells. The transfer requires Ca2+ or spermine, and is dependent on time, temperature, the concentration and composition of the vesicles, and the nature of the cellular lipopolysaccharide. The process results in bulk transfer of vesicle lipids to the cells rather than reciprocal molecular exchange between vesicles and the outer membrane. All components of mixed lipid vesicles, including cholesteryl oleate and lipopolysaccharide, are transferred to the cells in a ratio similar to that of the donor vesicles. The properties of the transfer process are consistent with direct fusion of vesicles with the outer membrane of the cell.