On the mechanism of sphingomyelin interaction with solubilized membrane proteins

Studies on the high-affinity receptor for IgE from rat basophilic leukemia cells (RBL-2H3) have shown that the phospholipid sphingomyelin remains attached to the protein complex during washing of the affinity immobilized complex under solubilizing conditions. Here we extended these findings and compared the species distribution patterns in sphingomyelin and phosphatidylcholine of the receptor-bound lipids to those of the plasma membrane lipids. FC epsilon-receptor-bound sphingomyelin but not phosphatidylcholine was enriched in long-chain fatty acids. We then examined other membrane proteins with respect to sphingomyelin enrichment. RBL-2H3 cell surface proteins, immobilized on concanavalin A-Sepharose and washed under solubilizing conditions, also showed a two- to six-fold enrichment in the associated sphingomyelin. Similar observations were also derived from other cell types, such as the mouse fibroblast cell line A-9 and the pig kidney epithelial cell line PK-1. Since this has been observed in all the three cell sources, it was suggested that sphingomyelin enrichment in FC epsilon-receptor preparations, although reproducible, was not specific for this protein. That this phenomenon was not specific for a particular protein might also be concluded from experiments that have shown nonhomogenous distribution of sphingomyelin in protein-free lipid-detergent mixtures. These results are compatible with a model whereby the interaction between sphingomyelin and soluble membrane proteins results from preference to nonmicellar phases or to structures with extended hydrophobic domains, probably due to the imperfect fitness of the detergent micelles to properly accomodate these lipids. This feature makes long-chain sphingomyelin a plausible candidate for the lipid responsible for the stabilizing effect that crude lipid preparations exert on the structural and functional properties of some membrane protein, e.g., FC epsilon R.     

Membrane insertion and lipid-protein interactions of bovine seminal plasma protein PDC-109 investigated by spin-label electron spin resonance spectroscopy

The interaction of the major acidic bovine seminal plasma protein, PDC-109, with dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by spin-label electron spin resonance spectroscopy. Studies employing phosphatidylcholine spin labels, bearing the spin labels at different positions along the sn-2 acyl chain indicate that the protein penetrates into the hydrophobic interior of the membrane and interacts with the lipid acyl chains up to the 14th C atom. Binding of PDC-109 at high protein/lipid ratios (PDC-109:DMPC = 1:2, w/w) results in a considerable decrease in the chain segmental mobility of the lipid as seen by spin-label electron spin resonance spectroscopy. A further interesting new observation is that, at high concentrations, PDC-109 is capable of (partially) solubilizing DMPC bilayers. The selectivity of PDC-109 in its interaction with membrane lipids was investigated by using different spin-labeled phospholipid and steroid probes in the DMPC host membrane. These studies indicate that the protein exhibits highest selectivity for the choline phospholipids phosphatidylcholine and sphingomyelin under physiological conditions of pH and ionic strength. The selectivity for different lipids is in the following order: phosphatidylcholine approximately sphingomyelin > or = phosphatidic acid (pH 6.0) > phosphatidylglycerol approximately phosphatidylserine approximately and rostanol > phosphatidylethanolamine > or = N-acyl phosphatidylethanolamine > cholestane. Thus, the lipids bearing the phosphocholine moiety in the headgroup are clearly the lipids most strongly recognized by PDC-109. However, these studies demonstrate that this protein also recognizes other lipids such as phosphatidylglycerol and the sterol androstanol, albeit with somewhat reduced affinity.     

Incorporation of sphingomyelin increases thermostability of human erythrocyte membrane and resistance of erythrocytes against thermal hemolysis

Erythrocytes of various mammal species differ markedly in their resistance against thermally induced hemolysis that was assumed related to the different sphingomyelin content of their membranes (J. Therm. Biol. 18 (1993) 177). In this work, two choline-containing lipids, sphingomyelin and phosphatidylcholine, were incorporated into the membrane of human erythrocytes and the resulting effect on thermal resistance and membrane thermostability of these cells was studied measuring thermohemolysis upon exposure to constant temperature and electrolyte leakage during transient heating, respectively. While sphingomyelin increased thermal resistance by 56% and increased the inducing temperature T_m of electrolyte leakage by 1±0.2°C, phosphatidylcholine produced practically no effect. The results show that sphingomyelin alone stabilized the structure of plasma membrane providing thermal stability to membrane proteins and in turn to whole cells as well.     

Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane. II. Protein-mediated transfer of monosaccharides and anions

In order to investigate the influence of membrane lipids on transport via the protein domain of the erythrocyte membrane, a number of facilitated diffusion processes was studied by tracer flux techniques in whole cells after cleavage of up to 65% of the phosphatidylcholine or the sphingomyelin by phospholipase A₂ from Naja naja or bee venom, or by sphingomyelinase, respectively.The mediated fluxes of l-arabinose, which is transported by the glucose carrier, and of l-lactate, which uses a specific monocarboxylate carrier, were markedly inhibited by cleavage of either phosphatidylcholine or sphingomyelin. These phospholipid dependencies are in line with earlier data on cholesterol dependencies (Deuticke, B. (1977) Rev. Physiol. Biochem. Pharmacol. 78, 1–97). They can only in part be explained by changes of membrane fluidity. More specific interactions of the degradation products with the carrier proteins seem also to play a role.Sulfate and oxalate transfer, which proceed via the inorganic anion-exchange system, are essentially unaffected by cleavage of phosphatidylcholine and less sensitive to sphingomyelin cleavage than the two other processes. This also agrees with earlier data on cholesterol independency of sulfate transfer. The inorganic anion-exchange protein thus seems to be less dependent on the surrounding lipids in its conformation and its mode of action than the two other carriers.     

Organelle biogenesis and intracellular lipid transport in eukaryotes.

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.     

The determination of the specific activity of picomolar amounts of long-chain acylcarnitine esters

Measurement of the specific activity of cellular pools of long-chain acylcarnitines is complicated by interference of other labeled cellular lipids, especially phosphatidylcholine and sphingomyelin. To overcome these problems the lipid extract from rabbit aorta labeled with [1-¹⁴C]palmitate was treated with phospholipase C. Upon two-dimensional thin-layer chromatography, the long-chain acylcarnitines could be isolated in an area free of interfering radioactivity. Mobility of long-chain carnitines was inversely proportional to the fatty acid chain length. The amount of long-chain acylcarnitine was quantified from their carnitine content after alkaline hydrolysis using carnitine acetyltransferase.     

Use of a photoactivable GM1 ganglioside analogue to assess lipid distribution in caveolae bilayer

A new photoactivable, radioactive derivative of ganglioside GM1 has been utilized to assess lipid distribution in the caveolae bilayer, taking advantage of the ability of the glycolipid, endogenous or exogenously added, to concentrate within this membrane compartment and to crosslink neighboring molecules upon illumination. After insertion into A431 plasma membrane and photoactivation, a membrane-enriched and a detergent-resistant fraction, enriched in gangliosides, sphingomyelin and cholesterol, were isolated. While a few radioactive proteins were detected in the membrane-enriched fraction, only radioactive caveolin was detected in the detergent-resistant fraction, indicating at the same time the enrichment of this fraction in caveolae and the presence of ganglioside within this compartment. Among lipids, crosslinked phosphatidylcholine, sphingomyelin and cholesterol were detected in the membrane-enriched fraction, while only crosslinked sphingomyelin was detected in the detergent-resistant fraction. These results suggest the enrichment in sphingomyelin - along with ganglioside - within the outer leaflet, and the preferential localization of cholesterol within the endoplasmic leaflet, of the caveolae bilayer.     

Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [³H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([³H]SM-N₃). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [³H]SM-N₃. However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.     

Use of a photoactivable GM1 ganglioside analogue to assess lipid distribution in caveolae bilayer

A new photoactivable, radioactive derivative of ganglioside GM1 has been utilized to assess lipid distribution in the caveolae bilayer, taking advantage of the ability of the glycolipid, endogenous or exogenously added, to concentrate within this membrane compartment and to crosslink neighboring molecules upon illumination. After insertion into A431 plasma membrane and photoactivation, a membrane-enriched and a detergent-resistant fraction, enriched in gangliosides, sphingomyelin and cholesterol, were isolated. While a few radioactive proteins were detected in the membrane-enriched fraction, only radioactive caveolin was detected in the detergent-resistant fraction, indicating at the same time the enrichment of this fraction in caveolae and the presence of ganglioside within this compartment. Among lipids, crosslinked phosphatidylcholine, sphingomyelin and cholesterol were detected in the membrane-enriched fraction, while only crosslinked sphingomyelin was detected in the detergent-resistant fraction. These results suggest the enrichment in sphingomyelin—along with ganglioside—within the outer leaflet, and the preferential localization of cholesterol within the endoplasmic leaflet, of the caveolae bilayer.     

Interaction of the acetylcholine (nicotinic) receptor protein from Torpedo marmorata electric organ with monolayers of pure lipids

Membrane fragments rich in cholinergic (nicotinic) receptor protein were purified from the electric organ of Torpedo marmorata. Their lipid composition is essentially characterized by the prominence of cholesterol, phosphatidylethanolamine and phosphatidylcholine, long-chain fatty acyl constituents, and the absence of sphingomyelin. Solubilised receptor was purified from these fragments and the concentration of sodium cholate lowered by dialysis to 0.01% (w/v). When this preparation was injected under a lipid monolayer, an increase of surface pressure developed, which was not observed with the detergent alone nor in the absence of lipid film. When covalently radiolabelled receptor preparations were injected at a constant surface pressure the radioactivity recovered with the film was proportional to the increase in area. It is concluded that the pressure or area increases are due to the penetration of the cholinergic receptor protein into the lipid film. Incorporation experiments into films formed from various pure lipids showed that the protein interacts more readily with cholesterol than with ergosterol, phosphatidylcholine, or other phospholipids. Its affinity is also higher for long-chain phosphatidylcholines than for short-chain ones. The degree of unsaturation and fluidity of the 3-sn-phosphatidylcholine (lecithin) films are of secondary importance. Parallel experiments with covalently and non-covalently labelled receptor preparations showed that part of the protein recovered with the film lost its alpha-toxin binding ability during the penetration. Similar data were obtained with the receptor purified from Electrophorus electricus electric organ.     

Interaction between sphingomyelin and a cytolysin from the sea anemone Stoichactis helianthus

The cytolytic toxin from the sea anemone Stoichactis helianthus was inhibited up to 90–95% by suspensions of sphingomyelin but not by phosphatidylcholine or other membrane lipids. When the toxin was incubated with spingomyelin and the mixture fractionated either by isoelectric focusing or Sephadex gel filtration, the residual hemolytic units migrated together with the lipid and not as free toxin. Incubation with phosphatidylcholine, however, did not shift the toxin peak in either type of column.A toxin-ferritin conjugate retaining hemolytic activity was observed by negative staining to bind to liposomers prepared with sphingomyelin but not with liposomes containing phosphatidylcholine. The results provide evidence that the membrane binding site of the toxin is sphingomyelin.     

Selective toxin-lipid membrane interactions of natural, haemolytic Scyphozoan toxins analyzed by surface plasmon resonance

A comparison of the molecular interaction of natural Scyphozoan lysins with their bioactivity in a haemolytic assay was performed by establishing an efficient, automatable and reproducible procedure for the measurement of protein-membrane interactions. The toxin-membrane interactions were analyzed utilising a chip-based technology with immobilized liposomes as artificial cell membranes. The technique was established with streptolysin O as a cholesterol-selective model toxin and its cholesterol-selectivity has been proven. The haemolytic potency of protein fractions derived from the venom of the jellyfish Aurelia aurita and Cyanea capillata was tested and EC50 values of 35.3 μg/mL and 43.1 μg/mL against sheep and 13.5 μg/mL and 8.8 μg/mL against rabbit erythrocytes were measured. Cell membrane binding as a first step in the haemolytic process was analyzed using the Biacore^® technology. Major cell membrane lipids (cholesterol, sphingomyelin and phosphatidylcholine) were immobilized as pure liposomes and in binary mixtures. A preference for cholesterol and sphingomyelin of both jellyfish species was demonstrated. The specificity of the method was proven with a non-haemolytic A. aurita protein fraction that did not express a lipid binding. Additionally, an inactivated C. capillata lysine with negligible haemolytic activity showed a remaining but reduced adsorption onto lipid layers. The binding level of the lytic venom fraction of these dominant boreal jellyfish species increased as a function of protein concentration. The binding strength was expressed in RU50 values ranging from 12.4 μg/mL to 35.4 μg/mL, which were in the same order of magnitude as the EC50 values in the haemolytic assay.     

The nature and location of Acholeplasma laidlawii membrane proteins investigated by two-dimensional gel electrophoresis

The high resolution, two-dimensional electrophoresis system for the separation of proteins described by O'Farrell, (O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007–4021) has been modified for the separation of Acholeplasma laidlawii proteins.Reproducible protein patterns have been obtained from A. laidlawii cell, membrane and soluble protein preparations. The isoelectric focusing of membrane proteins was greatly improved by removing the bulk of the membrane lipid before solubilizing the protein.A. laidlawii peripheral membrane proteins were removed from the membrane by low ionic strength washing and by treatment with EDTA. The effect of an exhaustive EDTA treatment and a rapid, warm EDTA treatment were compared. By comparing the protein patterns obtained in these ways it was possible to distinguish two separate groups of peripheral membrane proteins and one integral membrane protein group. The peripheral membrane proteins which were removed from the membrane at low ionic strength (group I) were also insoluble in Triton X-100, whereas additional peripheral membrane proteins extractable by subsequent EDTA treatment (group II) were soluble in Triton X-100.Exterior-facing membrane proteins were distinguished from the interiorfacing ones by lactoperoxidase-catalyzed iodination of intact cells and membranes. Group I peripheral membrane proteins faced the cell interior whereas group II proteins faced the cell exterior. We counted approximately 320 individual whole cell proteins. Of these, about 140 were membrane associated and a maximum of 40 proteins were iodinated after iodinationg intact cells.A. laidlawii was also grown in the presence of NaH₂³²PO₄ and whole cell proteins were separated by two-dimensional gel electrophoresis. One membrane protein and two soluble proteins were labelled.     

Sphingolipid-enriched membrane domains from rat cerebellar granule cells differentiated in culture. A compositional study

Sphingolipid-enriched membrane domains, characterized by a particular protein and lipid composition, have been detected in a variety of cells. However, limited data are available concerning these domains in neuronal cells. We analyzed the lipid and protein composition of a sphingolipid-enriched membrane fraction prepared from primary rat cerebellar granule cells differentiated in culture. Although the protein content of this fraction was only 1.4% of total cellular protein, 60% of the gangliosides, 67% of the sphingomyelin, 50% of the ceramide, and 40% of the cholesterol were located in this fraction. The protein pattern of the sphingolipid-enriched domain fraction was dramatically different from that associated with the cell homogenate. This fraction contained 25% of the tyrosine-phosphorylated proteins and was enriched in two proteins with apparent molecular masses of 135 and 15 kDa. 12% of cellular glycerophospholipids were located in the fraction, with phosphatidylcholine having the highest enrichment. The molar ratio between proteins, glycerophospholipids, cholesterol, sphingomyelin, ceramide and gangliosides in cerebellar granule cells was 1.6:41.6:6. 1:1.3:0.3:1 in the cell homogenate and 0.04:8.3:4.0:1.4:0.2:1 in the sphingolipid-enriched membrane fraction. These data indicate that selected proteins segregate with sphingolipids in specialized domains in the membrane of cultured neurons.     

The Effect of Apical and Basolateral Lipids on the Function of the Band 3 Anion Exchange Protein

Although many polarized proteins are sorted to the same membrane domain in all epithelial tissues, there are some that exhibit a cell type–specific polarity. We recently found that band 3 (the anion exchanger AE1) was present in the apical membrane of a renal intercalated cell line when these cells were seeded at low density, but its targeting was reversed to the basolateral membrane under the influence of an extracellular matrix protein secreted when the cells were seeded at high density. Because apical and basolateral lipids differ in epithelia, we asked what effect might these lipids have on band 3 function. This question is especially interesting since apical anion exchange in these cells is resistant to disulfonic stilbene inhibitors while basolateral anion exchange is quite sensitive. Furthermore, the apical anion exchanger cannot be stained by antibodies that readily identify the basolateral protein.

We used short chain sphingolipid analogues and found that sphingomyelin was preferentially targeted to the basolateral domain in the intercalated cell line. The ganglioside GM₁ (Gal 1β1, 3GalNAcβ1, 4Gal-NeuAcα2, 3Galβ1, 4Glc ceramide) was confined to the apical membrane as visualized by confocal microscopy after addition of fluorescent cholera toxin to filter grown cells. We reconstituted erythrocyte band 3 into liposomes using apical and basolateral types of lipids and examined the inhibitory potency of 4,4′-dinitorsostilbene-2,2′-disulfonic acid (DNDS; a reversible stilbene) on ³⁵SO₄/SO₄ exchange. Although anion exchange in sphingomyelin liposomes was sensitive to inhibition, the addition of increasing amounts of the ganglioside GM₁ reduced the potency of the inhibitor drastically. Because these polarized lipids are present in the exofacial surface of the bilayer, we propose that the lipid structure might influence the packing of the transmembrane domains of band 3 in that region, altering the binding of the stilbenes to these chains. These results highlight the role of polarized lipids in changing the function of unpolarized proteins or of proteins whose locations differ in different epithelia.

     

Bicarbonate/chloride transport kinetics at 37°C and its relationship to membrane lipids in mammalian erythrocytes

The rate of exchange of HCO₃^? with Cl^? at 37°C in erythrocytes of ten mammalian species was studied. The rate constant increases from 7s^?1 (ox) to 16s^?1 (rat), and is inversely proportional to the body size (log₁₀) of the species. It is found that the membrane permeability in different species is positively correlated to the red cell membrane phosphatidylcholine or arachidonate content, and is negatively correlated to the sphingomyelin or linoleate content.     

Lipid transfer proteins in the study of artificial and natural membranes

Summary Lipid transfer proteins, differing in their specificity for the transfer of lipids and for the surfaces on which they act, have been purified from various mammalian tissues and subsequently characterized. Several of their properties make them useful research tools. They have been used alone or with other techniques to study the distribution and mobility of phospholipids in artificial vesicles and in natural membranes, and have been used to create asymmetric phospholipid vesicles.Lipid transfer proteins are capable of altering the lipid composition of membranes by introducing new lipids or by depletion of existing lipids. Some of the transfer proteins can effect a net transfer of phospholipids, glycosphingolipids and cholesterol from one structure to another, whereas others appear to act primarily in promoting exchange. Some lipid transfer proteins are capable of introducing spin labeled and fluorescent lipid analogs into the outer surface of membranes. Because lipid transfer proteins do not seem to alter membrane lipid asymmetry or permeability of membranes, they are useful tools for studying the effect of lipid substitution on membrane-mediated transport processes and on various membrane-bound enzyme systems.Abbreviations PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PG phosphatidylglycerol - PS phosphatidylserine - DPG diphosphatidylglycerol - SPH sphingomyelin - G_{m t} II³--N-Acetylneuraminosylgangliotetraglycosylceramide - GbOse₄Cer globotetraglycosylceramide Career Investigator of C.O.N.I.C.E.T. (Argentina)Career Investigator of the American Heart Association.     

Isolation and biochemical characterisation of lipid rafts from Atlantic cod (Gadus morhua) intestinal enterocytes

Lipid rafts are glycosphingolipid/cholesterol-enriched membrane microdomains that have been extensively studied during the past two decades. Our aim was to isolate and perform biochemical characterization of lipid rafts from the intestinal brush border membrane (BBM) of Atlantic cod (Gadus morhua) to confirm their existence in a cold-water species and compare their characteristics with lipid rafts from other species in terms of lipid and protein content. To validate the isolation process, we assayed marker enzymes for subcellular organelles, including alkaline phosphatase (AP) and leucine aminopeptidase (LAP), both well-known marker enzymes for BBM and lipid rafts. All biochemical methods showed enrichment of AP in both the BBM and lipid raft fractions. Proteomic studies were performed by MALDI-TOF mass spectrometry using trypsin digested SDS-PAGE samples. Various proteins were associated with the cod intestinal lipid raft preparation such as aminopeptidase-N, prohibitin, and beta-actin. Lipid analysis with ³¹P NMR and thin layer chromatography on BBMs and lipid rafts samples gave higher content of sphingomyelin than previously reported in the BBM and lower content of phosphatidylcholine. Furthermore, sphingomyelin was highly dominant in the lipid rafts together with cholesterol. The existence of lipid rafts containing previously reported lipid raft characteristics from the cod intestine has, therefore, been confirmed in a ray-finned fish for the first time to the best of our knowledge.     

Lateral segregation of membrane lipids and formation of stable rod-shaped membrane projections in erythrocytes treated with long-chain alcohols

Human erythrocytes, incubated with sonicated dispersions of phosphatidylcholine, cholesterol and saturated straight-chain alcohols (C₁₆-C₁₈) develop stiff, rod-shaped, hemoglobin-containing membrane projections within 120 min. The number of these ‘rods’ varies (1–3 per cell), they reach a length of up to 14 μm (twice the cell diameter) and a thickness of 0.3–1.0 μm. ‘Rods’ may be separated from ‘residual cells’ by shear flow and centrifugation without severe hemolysis. Lipid analyses carried out on residual cells and rods indicate lateral segregation of the phospholipids of the outer leaf of the membrane lipid bilayer (phosphatidylcholine and sphingomyelin) and of the alcohol applied. Phosphatidylcholine accumulates in the residual cells, sphingomyelin and the alcohol in the rods. No differences in membrane protein patterns were observed between rods and residual cells. The rod-shape is dependent on the presence of the alcohol, extraction of the alcohol converts rods into hemoglobin-containing spheres without lysis. The formation of rods, which is indicative of a lateral phase separation, is discussed in terms of lipid-lipid interactions and with respect to parameters determining the shape of cells.     

Lipids of rabies virus and BHK-21 cell membranes.

The lipid composition of highly purified Flury strain of rabies virus (HEP) propagated in BHK-21 cells in a chemically defined medium was observed to be 6.7% neutral lipids, 15.8% phospholipids, and 1.5% glycolipids. In the virion, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the most abundant phospholipids, accounting for 90% of the total, and the molar ratio of cholesterol to phospholipid was 0.48. Uninfected BHK-21 cell membranes were obtained by nitrogen cavitation techniques and separated by density gradient centrifugation, and the membranes were assayed for purity using 5'-nucleotidase, cytochrome oxidase, and reduced nicotinamide adenine dinucleotide phosphate diaphorase activities. Lipids of the plasma membrane were enriched in cholesterol, phosphatidylcholine, and phosphatidylethanolamine. In contrast, membranes of the endoplasmic reticulum were enriched in phosphatidylcholine, but contained smaller amounts of phosphatidylethanolamine and sphingomyelin. Comparison of the fatty acyl chains of virus and membranes from uninfected cells revealed the virion to have the lowest ratio of C18:1 to C18:0 (1.771), compared with values of about 3.0 for the plasma membrane and endoplasmic reticulum. Total polyenoic fatty acids were enriched in the plasma membrane, whereas the virus contained higher amounts of total saturates than either of the two membrane preparations. Analysis of the polar and neutral lipid fractions as well as the acyl chain analysis suggests the virion has a lipid composition that is intermiediate to that of the plasma membrane and endoplasmic reticulum and is consistent with the view that numerous viral particles are synthesized de novo by not utilizing a preexisting membrane template. From the ratio of cholesterol to phospholipid of 0.48, we calculated that 1.92 X 10(5) molecules of lipid would cover 4.14 X 10(4) nm2 in the form of a bilayer. Considerations of the molecular dimensions of the rabies envelope (total surface area, 5 X 10(4) nm2) as a bilayer suggest that some penetration of lipids by envelope proteins (M and G) is necessary.