Different sphingolipids show differential partitioning into sphingolipid/cholesterol-rich domains in lipid bilayers

Two fluorescence-based approaches have been applied to examine the differential partitioning of fluorescent phospho- and sphingolipid molecules into sphingolipid-enriched domains modeling membrane "lipid rafts." Fluorescence-quenching measurements reveal that N-(diphenylhexatrienyl)propionyl- (DPH3:0-)-labeled gluco- and galactocerebroside partition into sphingolipid-enriched domains in sphingolipid/phosphatidylcholine/cholesterol bilayers with substantially higher affinity than do analogous sphingomyelin, ceramide, or phosphatidylcholine molecules. By contrast, the affinity of sphingomyelin and ceramide for such domains is only marginally greater than that of a phosphatidylcholine with similar hydrocarbon chains. By using direct measurements of molecular partitioning between vesicles of different compositions, we show that the relative affinities of different C(6)-NBD- and C(5)-Bodipy-labeled sphingolipids for sphingolipid-enriched domains are quantitatively, and in most circumstances even qualitatively, quite different from those found for species whose N-acyl chains more closely resemble the long saturated chains of cellular sphingolipids. These findings lend support in principle to previous suggestions that differential partitioning of different sphingolipids into "raft" domains could contribute to the differential trafficking of these species in eukaryotic cells. However, our findings also indicate that short-chain sphingolipid probes previously used to examine this phenomenon are in general ill-suited for such applications.     

Comparative dynamics and location of chain spin-labelled sphingomyelin and phosphatidylcholine in dimyristoyl phosphatidylcholine membranes studied by EPR spectroscopy

The dynamics and environment of sphingomyelin spin-labelled at different positions in the N-acyl chain have been studied in dimyristoyl phosphatidylcholine bilayer membranes by using electron spin resonance spectroscopy. Comparison was made with phosphatidylcholine spin-labelled on the sn-2 acyl chain in the same host membrane. Spin-labelled sphingomyelin was found to mix well with the host phosphatidylcholine lipids in both gel and fluid phase membranes. At 1 mol%, mutual spin-spin interactions are no greater than for spin-labelled phosphatidylcholine. In the fluid membrane phase, the effective chain order parameters and polarity-sensitive isotropic hyperfine coupling constants of spin-labelled sphingomyelin display a similar dependence on the position of labelling to those of spin-labelled phosphatidylcholine. The values of both parameters are, however, generally larger for sphingomyelin than for phosphatidylcholine at equivalent positions of acyl chain labelling. This difference is attributed to the different chain linkage of sphingo- and glycero-lipids, combined with an offset of approximately one C-atom in transbilayer register between the respective N-acyl and O-acyl chains. In the gel phase, differences in chain configuration between sphingomyelin and phosphatidylcholine are indicated by differences in spin label spectral anisotropy between the two lipids, which appears to reverse towards the terminal methyl chain end.     

Spin-label electron spin resonance studies on the mode of anchoring and vertical location of the N-acyl chain in N-acylphosphatidylethanolamines

Electron spin resonance (ESR) studies have been performed on N-myristoyl dimyristoylphosphatidylethanolamine (N-14-DMPE) membranes using both phosphatidylcholines spin-labeled at different positions in the sn-2 acyl chain and N-acyl phosphatidylethanolamines spin-labeled in the N-acyl chain to characterize the location and mobility of the N-acyl chain in the lipid membranes. Comparison of the positional dependences of the spectral data for the two series of spin-labeled lipids suggests that the N-acyl chain is positioned at approximately the same level as the sn-2 chain of the phosphatidylcholine spin-label. Further, similar conclusions are reached when the ESR spectra of the N-acyl PE spin-labels in dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylethanolamine (DMPE) host matrixes are compared with those of phosphatidylcholine spin-labels in these two lipids. Finally, the chain ordering effect of cholesterol has also been found to be similar for the N-acyl PE spin-label and PC spin-labels, when the host matrix is either DMPC and cholesterol or N-14-DMPE and cholesterol at a 6:4 mole ratio. In both cases, the gel-to-liquid crystalline phase transition is completely abolished but cholesterol perturbs the gel-phase mobility of N-14-DMPE more readily than that of DMPC. These results demonstrate that the long N-acyl chains are anchored firmly in the hydrophobic interior of the membrane, in an orientation that is parallel to that of the O-acyl chains, and are located at nearly the same vertical position as that of the sn-2 acyl chains in the lipid bilayer. There is a high degree of dynamic compatibility between the N-acyl chains and the O-acyl chains of the lipid bilayer core, although bilayers of N-acyl phosphatidylethanolamines possess a more hydrophobic interior than phosphatidylcholine bilayers. These results provide a structural basis for rationalizing the biological properties of NAPEs.     

Phospholipid substitution of the purple membrane. The stoichiometry of light-induced proton release by phospholipid-substituted purple membranes

The method of Warren et al. (1974, Proc. Natl. Acad. Sci. U.S. 71, 622–626) was employed to substitute the polar lipids of the purple membrane of Halobacterium halobium by different phosphatidylcholine species. Substitution at pH 6.5 yields proteolipid complexes in the form of bent open sheets which have a protein to lipid phosphorus ratio similar to the natural membrane, i.e. about 1 : 10 (mol/mol). The extent of substitution increases with the length of the fatty acid chain of the phosphatidylcholine used.The spectral properties of bacteriorhodopsin are only slightly affected by substitution of 95% of the lipid, except that the photocycle is slowed down appreciably. Due to this slow rate the M_{4 12} intermediate of the cycle accumulates in the light. Associated with this accumulation is a net light-induced proton release, which proved insensitive to uncoupler. A comparison between the net proton release and the amount of M_{4 12} accumulated, studied as a function of pH, shows that no fixed stoichiometry exists between the two processes.Phospholipid substitution by egg phosphatidylcholine at pH 7.5 or by egg phosphatidylethanolamine leads to preparations of purple membrane with 15 or 25 mol of phospholipid per mol of bacteriorhodopsin, respectively. These preparations seem to consist of closed membrane structures. They take up protons in the light in an uncoupler-sensitive way.     

Capillary electrophoretic enzyme immunoassay with electrochemical detection for thyroxine

A capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) has been developed. In this method, antigen (Ag) competes with horseradish peroxidase (HRP)-labeled antigen (HRP-Ag) for a limited number of antibody (Ab) binding sites. The free HRP-Ag and the bound HRP-Ag-Ab complex are separated by capillary electrophoresis in a separation capillary. Then they catalyze the oxidation of their enzyme substrate 3,3',5,5'-tetramethylbenzide (TMB (reduced form)) with H(2)O(2) in a reaction capillary, which follows the separation capillary. The reaction product (TMB (oxidized form)) is amperometrically determined using a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Due to the amplification of the enzyme, the concentration of TMB(Ox) is much higher than those of free HRP-Ag and the bound HRP-Ag-Ab complex. Therefore, the limit of detection (LOD) of CE-EIA-ED is very low. The method has been used to determine thyroxine in human serum. A concentration of LOD of 3.8 x 10(-9)mol/L, which corresponds to a mass LOD of 23.2 amol, was achieved.     

Cell-free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane

NMR structure determination of large membrane proteins is hampered by broad spectral lines, overlap, and ambiguity of signal assignment. Chemical shift and NOE assignment can be facilitated by amino acid selective isotope labeling in cell-free protein synthesis system. However, many biological detergents are incompatible with the cell-free synthesis, and membrane proteins often have to be synthesized in an insoluble form. We report cell-free synthesis of subunits a and c of the proton channel of Escherichia coli ATP synthase in a soluble form in a mixture of phosphatidylcholine derivatives. In comparison, subunit a was purified from the cell-free system and from the bacterial cell membranes. NMR spectra of both preparations were similar, indicating that our procedure for cell-free synthesis produces protein structurally similar to that prepared from the cell membranes.     

Capillary electrophoretic enzyme immunoassay with electrochemical detection using a noncompetitive format

A capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) using a noncompetitive format has been developed. In this method, antigen (Ag) reacts with an excess amount of horseradish peroxidase (HRP)-labeled antibody (Ab*). The free Ab* and the bound Ag-Ab* complex produced in the solution are separated by capillary zone electrophoresis in a separation capillary. Then they catalyze enzyme substrate 3,3',5,5'-tetramethylbenzide (TMB(Red)) and H(2)O(2) in a reaction capillary following the separation capillary. The reaction product, TMB(Ox), can be determined using amperometric detection on a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Due to the amplification of the enzyme, a significant amount of TMB(Ox) can be produced for detection. Therefore, the limit of detection (LOD) of CE-EIA-ED is very low. A tumor marker (CA15-3) was used as a model, in order to test the method. The concentration LOD of CA15-3 is 0.024 U/ml, which corresponds to a mass detection limit of 1.3x10(-7) U.     

A 2H-NMR study on the interactions of the local anesthetic tetracaine with membranes containing phosphatidylserine

The interaction of the local anesthetic tetracaine with phosphatidylserine-containing model membranes has been studied by 2H-NMR. Charged tetracaine exhibited an unusually large partition coefficient into multilamellar dispersions of phosphatidylserine. The 2H-NMR spectra consisted of a Pake doublet and a narrow line, with the former corresponding to tetracaine in the bilayer and the latter to tetracaine free in solution. A strong pH dependence of the quadrupole splittings indicated different membrane locations for charged and uncharged tetracaine. In equimolar mixtures of phosphatidylserine and phosphatidylcholine the partition coefficients and 2H-NMR spectra were much more like those observed in neat phosphatidylcholine than in neat phosphatidylserine. Dilution studies at pH 5.5 indicated that in phosphatidylserine/phosphatidylcholine mixtures tetracaine experiences a three-site exchange similar to that found earlier for tetracaine in phosphatidylcholine. Tetracaine is in fast exchange between sites weakly bound to membrane and free in solution, and in slow exchange with a strongly bound site in the membrane.     

Photoinduced interaction of riboflavin dye with different reducing agents in aqueous and liposome media

The photoelectrochemical and spectral studies of riboflavin have been carried out in aqueous and phosphatidylcholine (PC) liposome media in presence of different reducing agents such as I-, Br-, Cl-, Fe2+, Fe(CN)6(4-) and Cu+. The results from both the studies support the photoinduced electron transfer from the reducing agent to the excited riboflavin dye. Moreover, a good correlation between photovoltages/Stern-Volmer quenching constants versus reduction potentials of the reducing agents also confirms the above electron transfer in the photoexcited state. An alternative method has been developed to determine the Stern-Volmer quenching constant.     

Membrane structure and dynamics by 2H- and 31P-NMR. Effects of amphipatic peptidic toxins on phospholipid and biological membranes

The actions of bee venom melittin and delta-lysin from Staphylococcus aureus on membranes have been monitored by solid-state deuterium and phosphorus NMR and shown to differ depending on temperature and on the lipid-to-peptide molar ratio Ri. In the gel phase of phosphatidylcholine model membranes, for lipid-to-peptide ratios Ri greater than 15, melittin induces isotropic lines interpreted as reflecting the presence of small discoidal structures, whereas delta-lysin does not. These small objects are metastable, that is, within a time-scale of hours they return to large lipid bilayers. The kinetics of this process depend on the lecithin chain length. In the fluid phases, at temperatures greater than that of the gel-to-fluid transition Tc, analysis of the quadruplar splittings in terms of chain ordering indicates that both melittin and delta-lysin similarly disorder the membrane. At temperatures above but close to Tc, melittin preferentially orders the center of the bilayer, while delta-lysin promotes ordering throughout the entire bilayer thickness. These effects are interpreted as reflecting different locations of the peptides with respect to the membrane surface. The addition of greater amounts of toxins, Ri = 4, on phosphatidylcholine model membranes induces very small structures irrespective of the temperature in the case of melittin, but only above Tc for delta-lysin. NMR spectral features similar to those characterizing the small fast-tumbling objects with phosphatidylcholine are also observed with egg phosphatidylethanolamine and erythrocyte membranes. The formation of small structures is thus inferred as a general process which reflects membrane supramolecular reorganization.(ABSTRACT TRUNCATED AT 250 WORDS)     

PMR relaxation times of micelles of the nonionic surfactant Triton X-100 and mixed micelles with phospholipids.

Previous pmr studies at 220 MHz have led to the suggestion that phosphatidylcholine and the nonionic surfactant Trition-X-100 form mixed micellar structures at high molar ratios of trition to phosphalipid. These mixed micelles provide one form of the phospholipid which the enzyme phospholipase A2 can utilize as substrate. Spin-lattice relaxation times (T1) and spin-spin relaxation times (T2) obtained from line widths for resolvable protons in Triton X-100 micelles and mixed micelles with egg phosphatidycholine and dipalmitoyl phosphatidylcholine are reported. They suggest that the structure of the mixed micelles is generally similar to that of pure Triton X-100 micelles. The T1 values for the phsopholipid in the mixed micelles are found to be similar to those reported for phospholipid in sonicated vesicle preparations which are used as membrane models, but the lines are somewhat sharper suggesting the possibility of less anisotropic motion in the mixed micelles than in the vesicles.     

Determination of the structural domain of ApoAI recognized by high density lipoprotein receptors.

There is good evidence that high density lipoprotein (HDL) interacts with high affinity sites present on hepatocytes. The precise nature of the ligand recognized by putative HDL receptors remains controversial, although there is a consensus that apolipoprotein AI (apoAI) is involved. This suggestion would be strengthened if a biologically active site demonstrating a high affinity for the receptor could be isolated. Cyanogen bromide fragments (CF) of apoAI (CF1-CF4) were complexed with phospholipid, and their ability to associate with the receptor was compared in various binding studies. Careful analysis of the concentration-dependent association of 125I-labeled dimyristoyl phosphatidylcholine (DMPC) recombinants to rat liver plasma membranes revealed high and low affinity binding components. As all DMPC recombinants displayed the low affinity binding component, it was postulated that this interaction was independent of the protein present in the particle and may well represent a lipid-lipid or lipid-protein association with the membranes. Only 125I-labeled CF4.DMPC displayed a high affinity binding component with similar Kd and Bmax (8 x 10(-9) M, 1.6 x 10(-12) mol/mg plasma membrane protein) to that of 125I-labeled AI.DMPC (7 x 10(-9), 1.4 x 10(-12) mol/mg plasma membrane protein). Similarly, egg yolk phosphatidylcholine complexes containing CF4 (CF4.egg PC) showed higher affinity binding than CF1-egg yolk phosphatidylcholine complexes confirming the results obtained with DMPC complexes. Furthermore, ligand blotting studies showed that only 125I-labeled CF4.DMPC associated specifically with HB1 and HB2, two HDL binding proteins recently identified in rat liver plasma membranes. We conclude that a region within the carboxyl-terminus of apoAI is responsible for the interaction with putative HDL receptors present in rat liver plasma membranes.     

Lem3p is essential for the uptake and potency of alkylphosphocholine drugs,edelfosine and miltefosine

The alkylphosphocholine class of drugs, including edelfosine and miltefosine, has recently shown promise in the treatment of protozoal and fungal diseases, most notably, leishmaniasis. One of the major barriers to successful treatment of these infections is the development of drug resistance. To understand better the mechanisms underlying the development of drug resistance, we performed a combined mutant selection and screen in Saccharomyces cerevisiae, designed to identify genes that confer resistance to the alkylphosphocholine drugs by inhibiting their transport across the plasma membrane. Mutagenized cells were first selected for resistance to edelfosine, and the initial collection of mutants was screened a second time for defects in internalization of a short chain, fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD))-labeled phosphatidylcholine reporter. This approach identified mutations in a single gene, YNL323W/LEM3, that conferred resistance to alkylphosphocholine drugs and inhibited internalization of NBD-labeled phosphatidylcholine. Loss of YNL323W/LEM3 does not confer resistance to N-nitroquinilone N-oxide or ketoconazole and actually increases sensitivity to cycloheximide. The defect in internalization is specific to NBD-labeled phosphatidylcholine and phosphatidylethanolamine. Labeled phosphatidylserine is internalized at normal levels in lem3 strains. LEM3 is a member of an evolutionarily conserved family and has two homologues in S. cerevisiae. Single point mutations that produce resistance to alkylphosphocholine drugs and inhibition of NBD-labeled phosphatidylcholine internalization were identified in several highly conserved domains. These data demonstrate a requirement for Lem3p expression for normal phosphatidylcholine and alkylphosphocholine drug transport across the plasma membrane of yeast.     

Photophysics of thionine dye in aqueous and liposome media in presence of different reducing agents.

The photoelectrochemical and spectral (both absorption and fluorescence) studies of thionine, a cationic phenothiazine dye, have been carried out in aqueous and phosphatidylcholine liposome media in the presence of different reducing agents, such as I(-), Br(-), Cl(-) and Fe(2+). The results show that the photovoltage generation from photoelectrochemical studies and Stern-Volmer quenching constant studied by fluorescence quenching support the photoinduced electron transfer from the reducing agent to the singlet excited thionine dye. Moreover, a good correlation between photovoltages/Stern-Volmer quenching constants vs. reduction potentials of the reducing agents also confirms the above electron transfer in the photoexcited state.     

Design, synthesis, and characterization of bis-phosphatidylcholine, a mechanistic probe of phosphatidylcholine transfer protein catalytic activity

The design, synthesis, and characterization of 1-(17,18-dithiatetratriacontandioyl)-bis(2-hexadecanoyl-sn-glycero -3- phosphocholine) is described. Bis-phosphatidylcholine is a dimeric phospholipid comprised of two glycerophosphocholine groups linked together by a disulfide bond at the distal ends of the sn-1 fatty acyl chains. Electron microscopy and [14C]glucose trapping studies indicate that hydrated dispersions of bis-phosphatidylcholine form closed, spherical structures which have diameters in the range of 125-500 nm. Sensitivity to phospholipase hydrolysis suggests that this bipolar lipid is organized in a membrane such that the two polar head groups of the molecular are oriented at the same surface of the membrane. Using conditions in which bovine liver phosphatidylcholine transfer protein transfers both unsaturated and saturated diacyl phosphatidylcholines between fluid phosphatidylcholine vesicles, no transfer of the bipolar phospholipid is observed. The lack of activity toward bis-phosphatidylcholine suggests that this molecule may be a useful tool for elucidating the role of membrane phosphatidylcholine in the catalytic mechanism of the phosphatidylcholine transfer protein.     

Hypotonicity stimulates phosphatidylcholine hydrolysis and generates diacylglycerol in erythrocytes

Exposure of skate erythrocytes to hypotonic medium stimulates a rapid increase in levels of 1,2-diacylglycerol. Other treatments which produce cell swelling such as replacement of a portion of medium NaCl with the permeant solutes ethylene glycol or ammonium chloride also stimulate increases in diacylglycerol. Whereas the reduction of medium osmolarity to 460 mosm (from 940) stimulated a persistent diacylglycerol increase, the increase after reduction to 660 mosm was transient, peaking at 2.5 min and then slowly declining. This decline could be prevented by preincubation with the diacylglycerol kinase inhibitor R59022. To investigate the source of the increased diacylglycerol, the rate of incorporation of [32P]PO4 into each major phospholipid was measured. Reduction of osmolarity to 660 mosm stimulated the incorporation of phosphate into phosphatidylcholine markedly, with a smaller increase observed into phosphatidylinositol. To demonstrate phosphatidylcholine hydrolysis, erythrocytes were prelabeled with [32P]PO4. Subsequent exposure to hypotonic (660 mosm) medium stimulated a decrease in radioactivity in phosphatidylcholine and a large increase in radioactivity in phosphatidic acid. When stimulated in the presence of ethanol, 32PO4-labeled phosphatidylethanol was formed, suggesting activation of phospholipase D. In addition, the initial formation of 32PO4-labeled phosphatidic acid was not sensitive to inhibition of diacylglycerol kinase, supporting the role of direct activation of phospholipase D. These results indicate that hypotonicity and the accompanying cell swelling induce cell membrane phospholipid turnover, predominantly phosphatidylcholine, and production of the protein kinase C activator, diacylglycerol, which appears to occur via activation of phospholipase D.     

Modification of valinomycin-mediated bilayer membrane conductance by 4,5,6,7-tetrachloro-2-methylbenzimidazole

Summary The compound, 4,5,6,7-tetrachloro-2-methylbenzimidazole (TMB), has been found to markedly modify the steady-state valinomycin-mediated conductance of potassium (K⁺) ions through lipid bilayer membranes. TMB alone does not contribute significantly to membrane conductance, being electrically neutral in solution. In one of two classes of experiments (I), valinomycin is first added to the aqueous phases then changes of membrane conductance accompanying stepwise addition of TMB to the water are measured. In a second class of experiments (II), valinomycin is added to the membrane-forming solution, follwed by TMB additions to the surrounding water. In both cases membrane conductance shows an initial increase with increasing TMB concentration which is more pronounced at lower K⁺ ion concentration. At TMB concentrations in excess of 10^–5 m, membrane conductance becomes independent of K⁺ ion concentration, in contrast to the linear dependence observed at TMB concentrations below 10^–7 m. This transition is accompanied by a change of high field current-voltage characteristics from superlinear (or weakly sublinear) to a strongly sublinear form. All of these observations may be correlated by the kinetic model for carriermedicated transport proposed by Läuger and Stark (Biochim. Biophys. Acta 211:458, 1970) from which it may be concluded that valinomycin-mediated ion transport is limited by back diffusion of the uncomplexed carrier at high TMB concentrations. Experiments of class I reveal a sharp drop of conductance at high (>10^–5 m) TMB concentration, not seen in class II experiments, which is attributed to blocked entry of uncomplexed carrier from the aqueous phases. Valinomycin initially in the membrane is removed by lateral diffusion to the surrounding torus. The time dependence of this removal has been studied in a separate series of experiments, leading to a measured coefficient of lateral diffusion for valinomycin of 5×10^–6 cm²/sec at 25°C. This value is about two orders of magnitude larger than the corresponding coefficient for transmembrane carrier diffusion, and provides further evidence for localization of valinomycin in the membrane/solution interfaces.     

Lipid asymmetry in rabbit small intestinal brush border membrane as probed by an intrinsic phospholipid exchange protein.

All classes of phospholipids present in brush border membrane are exchanged in a 1:1 ratio for egg phosphatidylcholine when brush border membrane vesicles from rabbit small intestine are incubated with small unilamellar vesicles of egg phosphatidylcholine. The exchange reaction exhibits biphasic kinetics similar to those of the hydrolysis of brush border membrane phospholipids by phospholipase A2 and sphingomyelinase C. In both reactions there is an initial fast phase followed by a markedly slower one. The phospholipid exchange appears to be catalyzed by intrinsic brush border membrane protein(s), while the digestion by phospholipases is mediated by externally added enzymes. From a comparison of the kinetics of phospholipid exchange and phospholipid hydrolysis, the following conclusions can be drawn: Both sets of experiments indicate the presence of two phospholipid pools differing in the rate of phospholipid exchange and hydrolysis. Except for sphingomyelin, the size of the two phospholipid pools derived from phospholipid exchange is in good agreement with that derived from phospholipid hydrolysis. This is the main finding of this work, and on the basis of this result the two lipid pools are tentatively assigned to phospholipid molecules located on the outer and inner layer of the brush border membrane. The slow rate of phospholipid exchange reflects the rate of transverse or flip-flop movement of phospholipids. The half-time of this motion is approximately 8 h for isoelectric (neutral) phospholipids such as phosphatidylethanolamine and approximately 80 h for negatively charged phosphatidylserine and phosphatidylinositol. Isoelectric phospholipids (phosphatidylcholine, phosphatidylethanolamine) are preferentially located on the inner (cytoplasmic) side (to about 70%) while the negatively charged phospholipids are more evenly distributed: 55-60% are located on the inner side.     

Biosynthesis of membrane lipids in rat axons

Compartmented cultures of sympathetic neurons from newborn rats were employed to test the hypothesis that the lipids required for maintenance and growth of axonal membranes must be synthesized in the cell body and transported to the axons. In compartmented cultures the distal axons grow into a compartment separate from that containing the cell bodies and proximal axons, in an environment free from other contaminating cells such as glial cells and fibroblasts. There is virtually no bulk flow of culture medium or small molecules between the cell body and axonal compartments. When [methyl-3H]choline was added to the cell body-containing compartment the biosynthesis of [3H]-labeled phosphatidylcholine and sphingomyelin occurred in that compartment, with a gradual transfer of lipids (less than 5% after 16 h) into the axonal compartment. Surprisingly, addition of [methyl-3H]choline to the compartment containing only the distal axons resulted in the rapid incorporation of label into phosphatidylcholine and sphingomyelin in that compartment. Little retrograde transport of labeled phosphatidylcholine and sphingomyelin (less than 15%) into the cell body compartment occurred. Moreover, there was minimal transport of the aqueous precursors of these phospholipids (e.g., choline, phosphocholine and CDP-choline) between cell compartments. Similarly, when [3H]ethanolamine was used as a phospholipid precursor, the biosynthesis of phosphatidylethanolamine occurred in the pure axons, and approximately 10% of the phosphatidylethanolamine was converted into phosphatidylcholine. Experiments with [35S]methionine demonstrated that proteins were made in the cell bodies, but not in the axons. We conclude that axons of rat sympathetic neurons have the capacity to synthesize membrane phospholipids. Thus, a significant fraction of the phospholipids supplied to the membrane during axonal growth may be synthesized locally within the growing axon.     

Membrane permeability changes with vitamin A/vitamin E mixed bilayers

Vitamin A (all trans-retinol) enhances the permeability of egg phosphatidylcholine liposomes to glucose, urea, and erythritol while vitamin E (α-tocopherol) decreases permeability to the same solutes. Egg phosphatidylcholine bilayers containing both vitamin A and vitamin E are shown to have an altered permeability more similar to that affected by vitamin E alone. The membrane stabilizing effect of vitamin E appears dominant over the membrane destabilizing effect of vitamin A.