Translocation and turnover of phospholipid analogs in plasma membrane-derived vesicles from cell cultures

The time-dependent accumulation of phosphatidyldimethylethanolamine in formaldehyde-induced vesicles obtained from a somatic cell hybrid line was investigated. From a number of considerations including a two-fold enrichment of cholesterol and sphingomyelin it was concluded that these vesicles were derived from the cell plasma membrane.A progressive depletion of phosphatidylcholine, the major vesicle phospholipid, was observed in cells supplemented for various time periods with dimethylethanolamine. This depletion was accompanied by a concomitant increase in the amount of lipid analog.The time-dependent alteration of the phospholipid polar head group in intact cells was almost identical to that observed in isolated plasma membrane vesicles, suggesting a rapid equilibration of the de novo synthesized phospholipid with the cell surface compartment. From the initial velocity rate, the time required for the phosphatidylcholine pool to double was about 12 h.Agarose-linked phospholipase A₂ was used to measure the relative composition of choline- and dimethylethanolamine-phosphoglycerides in the outer surface of vesicles prepared from cells with different degrees of polar head group substitution. The gradual appearance of lysodimethylethanolamine lipid analog in vesicles treated with phospholipase A₂ suggested an asymmetric distribution of the phospholipid between the interior and the exterior part of the vesicle. This asymmetry was maximal up to about 4 h following the addition of dimethylethanolamine to the culture medium and was of a transient nature as the lipid analog accumulated on both sides of the plasma membrane. Based on these measurements a fast followed by a slow translocation component could be distinguished with apparent doubling times of 7 and 43 h for the lipid analog, respectively. As the analog becomes the predominant cellular phospholipid a significant increase in the vesicle lipid fluidity was measured.     

Study of intervesicular phospholipid exchange by NMR

NMR spectroscopy with the use of non-penetrating paramagnetic probes permits in situ determination of the composition of the outer surface of phospholipid vesicles. The method was employed to follow the phospholipid exchange between phosphatidylinositol and phosphatidylcholine vesicles induced by a postmicrosomal protein fraction from rat liver. The effects of these proteins on the lipid bilayer and the structure of the vesicles produced by exchange were studied.     

Exchangeability and rate of flip-flop of phosphatidylcholine in large unilamellar vesicles, cholate dialysis vesicles, and cytochrome oxidase vesicles.

Three model membrane systems have been characterized in terms of their interaction with phospholipid exchange proteins. Large unilamellar vesicles of phosphatidylcholine prepared by ether vaporization are shown to be homogeneous by gel filtration. Phospholipid exchange proteins from three sources are capable of catalyzing the rapid exchange of approximately half of the phospholipid from these vesicles. The remaining pool of radioactive phospholipid is virtually nonexchangeable (t1/2 of several days). Small unilamellar vesicles of phosphatidylcholine prepared by cholate dialysis also exhibit two pools of phospholipid (65% rapidly exchangable, 35% very slowly exchangeable) when incubated with beef liver phospholipid exchange protein. Cytochrome oxidase vesicles prepared both by a cholate dialysis method and by a direct incorporation method have been fractionated on a Ficoll discontinuous gradient, and tested for interaction with beef heart exchange protein. Two pools of phospholipid are once again observed (70% rapidly exchangable, 30% nonexchangeable), even for vesicles which have incorporated the transmembranous enzyme at a phospholipid to protein weight ratio of 2. The size of the rapidly exchangeable pool of phosphatidylcholine for each of the vesicle systems is consistent with the calculated fraction of phospholipid in the outer monolayer. The extremely slow rate of exchange of the second pool of the second pool of phospholipid reflects the virtual nonexistence of phospholipid flip-flop in any of these model membranes.     

Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes

The exchange of phosphatidylcholine between [³²P]phosphatidylcholine liposomes and unlabeled mitochondria was catalyzed by a purified phospholipid exchange protein from bovine heart cytosol. The loss of [³²P]phosphatidylcholine from the liposomes appeared to proceed in two stages: with 100 units of phospholipid exchange protein per ml the half-time of initial stage was about 10 min and that of the final stage 4 days or greater. Agarose-gel chromatography of the liposomes showed an elution compatible with a homogeneous pool of small single walled vesicles. Treatment of phosphatidyl [¹⁴C]choline liposomes with phospholipase D (phosphatidylcholine phosphatidohydrolase) showed that labeled phospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholipase, but that the labeled phospholipid left after the rapid exchange was completed could not be hydrolyzed by phospholipase D. It is proposed that the rapidly exchanging phosphatidylcholine constitutes the outer layer of the liposome bilayer. The long half-lives of 4 days or more probably represent the transposition of Phosphatidylcholine from the inner to the outer layer of the liposome bilayer.     

The transbilayer movement of phosphatidylcholine in vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Vesicles have been prepared from 18 : 1_c/18 : 1_c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose, ³¹P-NMR and K⁺ trap measurements. Pools of phosphatidylcholine available for exchange have been investigated using phosphatidylcholine exchange protein from bovine liver. The protein-containing vesicles both exhibit exchangeable pools larger than the fraction of phosphatidylcholine in the outer monolayer, whereas in the protein-free vesicles the exchangeable pool is consistent with the outer monolayer. The results indicate that both glycophorin and the partially purified band 3 preparation enhance the transbilayer movement of phosphatidylcholine.     

Interactions of phospholipid vesicles with rat hepatocytes in vitro influence of vesicle-incorporated glycolipids

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either $\text{N-}\text{lignoceroyldihydrolactocerebroside}$ or the monosialoganglioside, G_M1, enhanced liposomal lipid uptake 4–5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin.In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [¹⁴C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%).The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryoleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicles is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.     

Utilization of diacylglycerol in phospholipid bilayers by pig brain diacylglycerol kinase and Rhizopus arrhizus lipase

Diacylglycerol kinase purified from pig brain cytosol could use sonication-dispersed diacylglycerol in the presence of its activator, phosphatidylcholine vesicles. However, the kinase failed to significantly use diacylglycerol cosonicated with phosphatidylcholine. Similarly, the kinase could not use diacylglycerol generated in microsomes by the back reaction of diacylglycerol choline phosphotransferase, though phospholipase C treatment of microsomes yielded effective substrate for the kinase. In order to elucidate the mechanism of these discrepant findings, we studied the activity of the purified kinase and Rhizopus arrhizus lipase utilizing dioleoylglycerol incorporated into various phospholipid vesicles. The inaccessibility of diacylglycerol contained in phospholipid vesicles was observed similarly for the two different enzymes. We considered that the apparent enzymic latency of diacylglycerol could be best accounted for by an extremely limited solubility of diacylglycerol in the outer leaflet of phospholipid bilayers. The experimental bases for this interpretation are: 1) diacylglycerol cosonicated with dihexanoyl phosphatidylcholine was exceptionally effective as substrate for the kinase; 2) the enzyme activities with cosonicated and separately sonicated lipids became similar when bile salts were present; 3) both enzymes could use diacylglycerol generated on phosphatidylcholine vesicles by a limited phospholipase C hydrolysis; and 4) phosphatidylcholine diacylglycerol vesicles at widely different molar ratios (from 1:0.014 to 1:0.2) were similarly ineffective as substrate for both enzymes.     

Effect of the phase transition on the transbilayer movement of dimyristoyl phosphatidylcholine in unilamellar vesicles

Dimyristoyl phosphatidylcholine rapidly exchanges between vesicles at 37°C without vesicle fusion.The rate of the transbilayer movement of dimyristoyl phosphatidylcholine in sonicated vesicles has been measured employing ¹³C NMR using N-¹³CH_3? labeled lipids which are introduced into the outer monolayer of non-labeled vesicles by a phosphatidylcholine exchange protein. The rate of transbilayer movement of dimyristoyl phosphatidylcholine shows a distinct maximum (halftime 4 h) in the temperature range at which the hydrocarbon phase transition occurs.The activation energy of the flip-flop rate above the phase transition is 23.7 ± 2.0 kcal/mol.     

Preparation of asymmetric,cerebroside sulfate-containing phospholipid vesicles

A procedure is described which inserts asymmetrically cerebroside sulfate (‘sulfatide’) into the outer leaflet of bilayered phospholipid vesicles. Cerebroside sulfate is adsorbed onto a cellulose, filter-paper support and, when incubated with phosphatidylcholine vesicles is transferred to and inserted into the outer leaflet of these vesicles. This transfer occurs at, or above the transition temperature of the phospholipid and follows a similar pattern with small or larger (‘fused’) unilamellar vesicles. The transfer is linear with time for 1–2 h and is maximal after about 6 h, when the sulfatide content reaches about 6 mol% of the total quantity of phospholipid, corresponding to about 10 mol% of the phospholipids present in the outer layer. Initial rates of sulfatide transfer were somewhat increased when the vesicles contained a positively charged lipid (e.g. stearylamine) and decreased when this lipid was negatively charged (e.g. dicetyl phosphate) or hydrophobic (e.g. cholesterol). Divalent ions markedly inhibited sulfatide transfer and monovalent ions did so to a lesser degree. Once incorporated into the outer leaflet of the vesicle, the sulfatide could not be removed by washing with buffer, 1 M NaCl or 1 M urea.     

Asymmetry of the site of choline incorporation into phosphatidylcholine of rat liver microsomes.

[14C]Choline was incorporated into microsomal membranes in vivo, and from CDP-[14C]choline in vitro, and the site of incorporation determined by hydrolysis of the outer leaflet of the membrane bilayer using phospholipase C from Clostridium welchii. Labelled phosphatidylcholine was found to be concentrated in the outer leaflet of the membrane bilayer with a specific activity approximately three times that of the inner leaflet. During incorporation of CDP-choline and treatment with phospholipase C the vesicles retained labelled-protein contents indicating that they remained intact. When the microsomes were opened with taurocholate after incorporation of [14C]choline in vivo, the labelled phosphatidylcholine behaved as a single pool. Selective hydrolysis of labelled phosphatidylcholine in intact vesicles is not, therefore, a consequence of specificity of phospholipase C. These results indicate that the phosphatidylcholine of the outer leaflet of the microsomal membrane bilayer is preferentially labelled by the choline-phosphotransferase pathway and that this pool of phospholipid does not equilibrate with that of the inner leaflet.     

1H-NMR study of the location and motion of ubiquinones in perdeuterated phosphatidylcholine bilayers

Ubiquinones (n = 1,2,3,4,7,9,10) and ubiquinols (n = 1,2,3,4,10) were incorporated into ordinary (protonated) or perdeuterated dimyristoyl phosphatidylcholine vesicles and were found to have significant local molecular motion. The motion of the quinone ring, as judged from the linewidth of the OCH₃ proton resonances, decreased in longer-chain ubiquinones. Minimum values for the transverse mobility (flip-flop rates) of ubiquinones-1,2,3,4,10, measured with the aid of lanthanide shift reagents, suggest that they are all able to function in a protonmotive ‘Q cycle’ during electron transport. As the length of the side chain increases beyond 1 isoprenoid unit, the quinone/quinol ring tends to be deeper in the outer monolayer of small sonicated vesicles and in both monolayers of larger freeze-thaw vesicles, but little or no change in depth is observed in the inner monolayer of small vesicles. The ubiquinol rings are closer to the membrane surface than are the ubiquinone rings. For side chain n = 9 or 10, a second resonance from the OCH₃ protons of ubiquinones and ubiquinols in vesicles appears in the ¹H-NMR spectrum. This is due to the presence of two types of vesicles with different ubiquinone/phospholipid ratios.     

Trans-bilayer pseudocontact shifts produced by lanthanide ions in the 1H and 31P-nmr spectra of phospholipid vesicular membranes and their temperature variation

1. Shifts in the ¹H and ³¹P-nmr signals originating from the outer and inner phosphorylcholine head-groups and from the lipid acyl chains are observed when phosphatidylcholine vesicles are treated with increasing extravesicular concentrations of the lanthanides Eu³⁺, Pr³⁺, Yb³⁺, and Dy³⁺. 2. The addition of KNCS to increase the binding of the lanthanide ions to the outer head-groups is used to demonstrate that the intravesicular group shifts are not caused by bulk susceptibility effects. 3. The magnitude and direction of the observed shifts in the ¹H-nmr spectrum are shown to be consistent with (a) pseudocontact interaction of the paramagnetic lanthanide ions with the outer phospholipid head-groups, (b) current views of the conformation of the phosphatidylcholine head-group in the presence of lanthanides, and (c) a conservation of magnetic field within the vesicles due to their spherical nature. 4. Variation of the shifts with temperature are compared for egg phosphatidylcholine and dipalmitoyl phosphatidylcholine. The temperature variation in shifts is also used to study phase transitions in each monolayer and phase separations in mixed lipid systems.     

Resistance of Ca2+-ATPase to dilution by excess phospholipid in reconstituted vesicles

Ca²⁺-ATPase and other membrane proteins of the sarcoplasmic reticulum membrane from rabbit skeletal muscle have been reconstituted into lipid vesicles with increasing amounts of phosphatidylcholine. The protein composition and phospholipid concentration of these vesicles were analyzed by determining the density of the reconstituted membrane vesicles on linear H₂O-²H₂O gradients, in a constant concentration of sucrose. In all combinations of the Ca²⁺-ATPase with a weight excess of phosphatidylcholine, the reconstituted vesicles had a phospholipid-to-protein ratio similar to that of the native sarcoplasmic reticulum membrane, even though both solubilization and mixing had occurred. These vesicles of low phospholipid and high protein content exhibited all the original Ca²⁺-ATPase activity and ATP-stimulated calcium transport. The Ca²⁺-ATPase, and the calcium-binding proteins to a lesser extent, may order the lipid in such a manner so as to maintain the initial stoichiometry of lipid to protein observed in the native sarcoplasmic reticulum membrane.     

The interaction of lanthanide and calcium salts with phospholipid bilayer vesicles: The validity of the nuclear magnetic resonance method for determination of vesicle bilayer phospholipid surface ratios

Contrary to a recent report (B. Sears et al., Biochemistry 15 (1976) 1635), it has been determined that the ratio of the number of phospholipids on the inner and outer surfaces of phospholipid bilayer vesicles can be accurately determined by NMR paramagnetic ion shift reagent studies of vesicles. It is concluded that the metal interacts with all of the phospholipid on the exposed bilayer surface. A ratio of outer phospholipid to inner surface phospholipid of 2.1 ± 0.1 is obtained regardless of the nucleus studies, position of the nucleus relative to the metal ion binding site, molar ratio of metal to phospholipid over three orders of magnitude, or location of the metal ion on the inside or outside of the vesicle. Additionally, P-31 NMR studies using LaCl₃ and CaCl₂ indicate that Ca²⁺ weakly interacts with egg PC vesicles and than the lanthanides are adequate substitutes for Ca²⁺ since neither metal is found to perturb measurably the average polar head group conformation.     

Cyclopropane fatty acid synthase of Escherichia coli. Stabilization, purification, and interaction with phospholipid vesicles.

The cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes the methylenation of the unsaturated moieties of phospholipids in a phospholipid bilayer. The methylene donor is S-adenosyl-L-methionine. The enzyme is loosely associated with the inner membrane of the bacterium and binds to and is stabilized by phospholipid vesicles. The enzyme has been purified over 500-fold by flotation with phospholipid vesicles and appears to be a monomeric protein having a molecular weight of about 90 000. The enzyme binds only to vesicles of phospholipids which contain either unsaturated or cyclopropane fatty acid moieties. CFA synthase is active on phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin, the major phospholipids of E. coli, and also has some activity on phosphatidylcholine. The enzyme is equally active on phospholipid vesicles in the ordered or the disordered states of the lipid phase transition. Studies with a reagent that reacts only with the phosphatidylethanolamine molecules of the outer leaflet of a phospholipid bilayer indicate that CFA synthase reacts with phosphatidylethanolamine molecules of both the outer and the inner leaflets of phospholipid vesicles.     

19F-NMR of fluorine labeled acyl chains in phospholipid bilayer vesicles

¹⁹F-labeled phospholipids, L-α-bis-(ω-fluoro palmitoyl)phosphatidylcholine, L-α-bis-(12,12-difluoro stearoyl)phosphatidylcholine and L-α-bis-(6,6-difluoro palmitoyl)phosphatidylcholine, were incorporated in phospholipid vesicles by sonication of aqueous lipid emulsions. Vesicles were prepared both from the pure fluorine substituted phospholipids as well as from lipid mixtures obtained by combining the fluorine substituted lipids with the synthetic phospholipids, L-α-dilauroyl-, L-α-dimyristoyl-, L-α-dipalmitoyl- and L-α-distearoyl-phosphatidylcholine. Characterization by gel permeation chromatography showed that stable unilamellar vesicles with diameters of ≌200 Å could be obtained with a minimum or absence of multilamellar material. The vesicles give rise to two ¹⁹F resonances in most cases as observed previously [K.J. Longmuir and F.W. Dahlquist, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 2716]. The chemical shift differences undergo systematic changes that confirm the interpretations that the dual ¹⁹F resonances arise from the inner and outer halves of the vesicle bilayer. The shift separation increases systematically as the fluorine label is positioned closer to the phospholipid headgroup and decreases systematically with increasing temperature. Both observations agree with what is currently known about phospholipid vesicle structure. Anomalous results are obtained with DSPC as host vesicle since only a single resonance of inbedded fluorinated phospholipids is found.     

Concerning the mechanism of action of bovine liver phospholipid exchange protein: Exchange or net transfer

Bovine liver phospholipid exchange protein catalyzes the transfer of phosphatidylcholine between donor and acceptor populations of single bilayer phospholipid vesicles. In comparing egg and dimyristoylphosphatidylcholine vesicles, larger transfer rates are found for the unsaturated phospholipid. The bidirectional transfer rates measured from donor to acceptor and from acceptor to donor, are equivalent, suggesting that the protein facilitates an exchange rather than a net transfer of phosphatidylcholine.     

Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids.

Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.     

High resolution proton magnetic resonance of sonicated phospholipids

We have recorded high resolution proton magnetic resonance spectra of sonicated phospholipid vesicles. The following lipids were used in separate experiments: phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine from egg yolk as well as dimyristoyl phosphatidylcholine. Mixed lipid vesicles were also investigated. Assignments of the peaks associated with the various protons of the different lipids are presented. It is shown that in favorable cases, it is possible to resolve the different phospholipid head groups of mixed lipid samples. Spin lattice relaxation times (T₁) of each peak were collected at 500 MHz and 90 MHz. The influence of the addition of a small concentration of spin labeled phospholipid on i) the linewidths ii) the spin lattice relaxation times, was determined. It is shown that nitroxide radicals selectively broaden the peaks associated with the protons localized at a comparable depth of the bilayer. On the other hand, T₁ are less selectively perturbed. Potential applicability of ¹H-NMR for the investigation of lipid-proton specificity in membranes is discussed.