Relationship between losses in cytochrome oxidase activity and peroxidation of monosaturated phosphatidylcholines and phosphatidylethanolamines.

Incubation of inner mitochondrial membranes from rat liver in the presence of inducers of peroxidation reactions, such as ascorbate or cysteine, produced a large loss in cytochrome oxidase activity parallel to the disappearance of phosphatidylcholine and phosphatidylethanolamine molecular species, which contained a saturated and an unsaturated fatty acid. The loss in enzyme activity was unrelated to alterations in other species of these phospholipids or other ones. These results may reflect the existence of specific associations within the membrane between cytochrome oxidase and monosaturated phosphatidylcholines and/or phosphatidylethanolamines.     

Fusion of Sindbis virus with model membranes containing phosphatidylethanolamine: implications for protein-induced membrane fusion

The pH-induced fusion of Sindbis virus with model lipid membranes containing phosphatidylethanolamine has been studied using a quantitative fluorescence technique. The headgroup and acyl chain domains of the lipids have been altered systematically to determine their effect on fusion. Unsaturated phosphatidylethanolamines (PE) have been found to promote fusion, either by themselves, or in combination with phosphatidylcholines (PC). Cholesterol added to a mixture of unsaturated PE and PC was also shown to increase the extent of viral fusion. The results of these studies have been interpreted in terms of a tentative model for the molecular aspects of the target membrane which are necessary for viral fusion. In this model, the target membrane must have a sufficiently-sized domain containing poorly hydrated lipids which are capable of existing in a non-bilayer arrangement.     

Membrane composition modulates the interaction between a new class of antineoplastic agents deriving from aromatic 2-chloroethylureas and lipid bilayers: a solid-state NMR study

This work presents the investigations of the interactions between nystatin, a polyene antibiotic, and phospholipids with various head groups (phosphatidylcholine and phosphatidylethanolamine) and acyl chains of different length and saturation degree. The experiments were performed with the Langmuir monolayer technique. Among phosphatidylethanolamines, DMPE, DPPE and DSPE were studied, while phosphatidylcholines were represented by DSPC and DOPC. The influence of the antibiotic on the molecular organization of the phospholipid monolayer was analysed with the compression modulus values, while the strength of nystatin/phospholipid interactions and the stability of the mixed monolayers were examined on the basis of the excess free energy of mixing values. The results obtained proved a high affinity of nystatin towards phospholipids. Nystatin was found to interact more strongly with phosphatidylcholines than with phosphatidylethanolamines. The most negative values of the excess free energy of mixing observed for the antibiotic and DOPC mixtures prove that nystatin favors the phospholipid with two unsaturated acyl chains. The results imply that nystatin/phospholipid interactions compete in the natural membrane with nystatin/sterol interactions, thereby affecting the antifungal activity of nystatin and its toxicity towards mammalian cells.     

Molecular analysis of the phospholipids of Escherichia coli k12

Phospholipids from Escherichia coli K12 were converted to 1,2-diacylglycerols with phospholipase C from Bacillus cereus. High-pressure liquid chromatography of 1,2-diacylglycerol p-methoxybenzoates on LiChrosorb RP-18 using 2-propanol/acetonitrile (35:65) as eluant permitted separation of 14 molecular species. The main combinations of fatty acids were 1-16:0-2-16:1, 1-16:0-2-cyclo-17:0 and 1-16:0-2-18:1. Positional isomers were not present. The 1,2-di-16:0 compound was present at a significant level (7-10 mol%). Proportions of molecular species varied between phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Phospholipid from the outer membrane of E. coli K12 contained a lower level of molecules with two unsaturated chains than was present in the cytoplasmic membrane. The method is sensitive, has good resolving power and employs readily available equipment.     

Defining raft domains in the plasma membrane

Many plasma membrane (PM) functions depend on the cholesterol concentration in the PM in strikingly nonlinear, cooperative ways: fully functional in the presence of physiological cholesterol levels (35~45 mol%), and nonfunctional below 25 mol% cholesterol; namely, still in the presence of high concentrations of cholesterol. This suggests the involvement of cholesterol‐based complexes/domains formed cooperatively. In this review, by examining the results obtained by using fluorescent lipid analogs and avoiding the trap of circular logic, often found in the raft literature, we point out the fundamental similarities of liquid‐ordered (Lo)‐phase domains in giant unilamellar vesicles, Lo‐phase‐like domains formed at lower temperatures in giant PM vesicles, and detergent‐resistant membranes: these domains are formed by cooperative interactions of cholesterol, saturated acyl chains, and unsaturated acyl chains, in the presence of >25 mol% cholesterol. The literature contains evidence, indicating that the domains formed by the same basic cooperative molecular interactions exist and play essential roles in signal transduction in the PM. Therefore, as a working definition, we propose that raft domains in the PM are liquid‐like molecular complexes/domains formed by cooperative interactions of cholesterol with saturated acyl chains as well as unsaturated acyl chains, due to saturated acyl chains' weak multiple accommodating interactions with cholesterol and cholesterol's low miscibility with unsaturated acyl chains and TM proteins. Molecules move within raft domains and exchange with those in the bulk PM. We provide a logically established collection of fluorescent lipid probes that preferentially partition into raft and non‐raft domains, as defined here, in the PM.     

Distribution of phospholipid molecular species in outer and cytoplasmic membrane of Escherichia coli.

The outer membrane of Escherichia coli K-12 contained a smaller proportion of phospholipid molecular species with two unsaturated fatty acyl chains than did the cytoplasmic membrane. Proportions of phospholipid molecular species in the outer and cytoplasmic membranes changed in response to temperature changes. As the temperature increased, the content of 1-palmitoyl-2-cis-9,10-methylenehexadecanoyl species increased. Translocation of phospholipids from the cytoplasmic membrane to the outer membrane and synthesis of various molecular species were observed.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 degrees C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus.

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the identity of the fatty acyl chains can uniquely specify or determine which side of the membrane individual phosphatidylethanolamine molecules come to occupy. Although fatty acyl chain asymmetry and phosphatidylethanolamine asymmetry are correlated in VSV, no simple rules can be discerned which uniquely relate the two paramaters.     

Docosahexaenoic acid affects cell signaling by altering lipid rafts

With 22 carbons and 6 double bonds docosahexaenoic acid (DHA) is the longest and most unsaturated fatty acid commonly found in membranes. It represents the extreme example of a class of important human health promoting agents known as omega-3 fatty acids. DHA is particularly abundant in retinal and brain tissue, often comprising about 50% of the membrane's total acyl chains. Inadequate amounts of DHA have been linked to a wide variety of abnormalities ranging from visual acuity and learning irregularities to depression and suicide. The molecular mode of action of DHA, while not yet understood, has been the focus of our research. Here we briefly summarize how DHA affects membrane physical properties with an emphasis on membrane signaling domains known as rafts. We report the uptake of DHA into brain phosphatidylethanolamines and the subsequent exclusion of cholesterol from the DHA-rich membranes. We also demonstrate that DHA-induced apoptosis in MDA-MB-231 breast cancer cells is associated with externalization of phosphatidylserine and membrane disruption ("blebbing"). We conclude with a proposal of how DHA incorporation into membranes may control cell biochemistry and physiology.     

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 °C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

The study on the interaction between phytosterols and phospholipids in model membranes

Sterols are one of the major components of cellular membranes. Although in mammalian membranes cholesterol is a predominant sterol, in the human organism plant sterols (phytosterols) can also be found. Phytosterols, especially if present in concentrations higher than normal (phytosterolemia), may strongly affect membrane properties. In this work, we studied phytosterol-phospholipid interactions in mixed Langmuir monolayers serving as model membranes. Investigated were two phytosterols, beta-sitosterol and stigmasterol and a variety of phospholipids, both phosphatidylethanolamines and phosphatidylcholines. The phospholipids had different polar heads, different length and saturation of their hydrocarbon chains. The interactions between molecules in mixed sterol/phospholipid films were characterized with the mean area per molecule (A(12)) and the excess free energy of mixing (DeltaG(Exc)). The effect of the sterols on the molecular organization of the phospholipid monolayers was analyzed based on the compression modulus values. It was found that the incorporation of the phytosterols into the phospholipid monolayers increased their condensation. The plant sterols revealed higher affinity towards phosphatidylcholines as compared to phosphatidylethanolamines. The phytosterols interacted more strongly with phospholipids possessing longer and saturated chains. Moreover, both the length and the saturation of the phosphatidylcholines influenced the stoichiometry of the most stable complexes. Our results, compared with those presented previously for cholesterol/phospholipid monolayers, allowed us to draw a conclusion that the structure of sterol (cholesterol, beta-sitosterol, stigmasterol) does not affect the stoichiometry of the most stable complexes formed with particular phospholipids, but influences their stability. Namely, the strongest interactions were found for cholesterol/phospholipids mixtures, while the weakest for mixed systems containing stigmasterol.     

NADH-cytochrome c reductase, succinate cytochrome c reductase and phospholipids.

Phospholipid peroxidation of isolated rat liver inner mitochondrial membranes induced by either ascorbate or cysteine was accompanied by a release of flavins and coenzyme Q. A straight correlation between this release and the alteration of molecular species of phosphatidylcholine and phosphatidylethanolamine containing one saturated and one unsaturated fatty acid has been found. Peroxidation induced on molecular species of phosphatidylcholine and phosphatidylethanolamine containing only unsaturated fatty acids were accompanied by losses in enzyme activities of NADH-cytochrome c reductase and succinate cytochrome c reductase.     

Comparative study of the lipid composition of rabbit and lobster sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit and lobster muscles have similar phospholipid classes, but they differ in plasmalogen content. The plasmalogenic species are mostly distributed among phosphatidylethanolamines (PE's) and make up about 62% of the total in rabbit SR and about 46% in lobster membranes. Lobster SR phospholipids contain large amounts of polyunsaturated fatty acids which are present in low amounts in rabbit membranes. The total unsaturated fatty acids of phosphatidylcholines (PC's) represent about 53% and 73% of the total fatty chains for rabbit and lobster SR, respectively. The values found for PE's were about 56% and 64%, respectively. Furthermore, lobster membranes contain significant amounts of PC and PE molecular species with unsaturated fatty acids in positions 1 and 2, whereas rabbit SR contain low amounts.     

Helicobacter pylori lipids can form ordered membrane domains (rafts)

Ordered lipid domains (rafts) are generally considered to be features of eukaryotic cells, but ordered lipid domains formed by cholesterol lipids have been identified in bacteria from the genus Borrelia, and similar cholesterol lipids exist in the bacterium Helicobacter pylori. To determine whether H. pylori lipids could form ordered membrane domains, we investigated domain formation in aqueous dispersions of H. pylori whole lipid extracts, individual H. pylori lipids, or defined mixtures of H. pylori lipids and other membrane-forming lipids. DPH (1,6-diphenyl-1,3,5-hexatriene) anisotropy measurements were used to assay membrane order and FRET (Förster resonance energy transfer) was used to detect the presence of co-existing ordered and disordered domains. We found that H. pylori membrane lipid extracts spontaneously formed lipid domains. Domain formation was more stable when lipids were extracted from H. pylori cells grown in the presence of cholesterol. Certain isolated H. pylori lipids (by themselves or when mixed with other lipids) also had the ability to form ordered domains. To be specific, H. pylori cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside (CAG) and cholesterol-6-O-phosphatidyl-α-D-glucopyranoside (CPG) had the ability to form and/or stabilize ordered domain formation, while H. pylori phosphatidylethanolamine did not, behaving similarly to unsaturated phosphatidylethanolamines. We conclude that specific H. pylori cholesterol lipids have a marked ability to form ordered lipid domains.     

Variation in corn (Zea mays L.) for fatty acid compositions of triglycerides and phospholipids

The percentage of linoleic acid in corn germ oil of three crosses, C103D × B73, C103D×B84, and T220×H51, and their reciprocals was investigated. Corn germ oil from F₂, F₃, and backcrossed generations was also examined. More than one gene locus appeared to be involved in conditioning the linoleic acid content in these crosses. Strong maternal effects were exhibited in the F₁'s. Genotype also superimposed variations in fatty acid compositions within the characteristic lipid class patterns of the phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Fatty acid placements in triglycerides, digalactosyldiglycerides, and phospholipids of one inbred, H51, were determined by lipase and phospholipase hydrolysis. The overall pattern of placement showed that the fatty acids at the 1 position were predominately saturated and those at the 2 position were predominately unsaturated, but the fatty acid distribution was different for each individual lipid class. The molecular species of the phosphatidylcholines and phosphatidylethanolamines were separated by silver nitrate thin-layer chromatography. The major differences in the molecular species were a higher level of the dienoic-dienoic species and a lower level of the monoenoic-monoenoic species in the phosphatidylethanolamines than in the phosphatidylcholines.     

Distribution of phospholipid molecular species containing arachidonic acid and cholesterol in V79-UF cells

V79-UF cells were isolated from Chinese hamster V79 cells as a cell line that requires exogenous unsaturated fatty acids for growth. V79-UF cells incorporated arachidonic acid into phospholipids. The molecular species of diacyl phosphatidylcholine and phosphatidylethanolamine containing arachidonic acid comprised 61.4 and 70.5% of the total phospholipid molecular species in total membranes and 58.1 and 64.7% in plasma membrane, respectively. Polyunsaturated molecular species were distributed in a higher amount in the intracellular membranes than in the plasma membrane. No significant difference was seen in the diffusion coefficient between the plasma membranes from cells supplemented with oleic and arachidonic acids in spite of a distinct difference in the degree of unsaturation between the molecular species of these plasma membranes. The amount of cholesterol in the plasma membrane was higher in the cells grown in the presence of arachidonic acid than in those grown in the presence of oleic acid.     

Vitamin E and its function in membranes

Vitamin E is a fat-soluble vitamin. It is comprised of a family of hydrocarbon compounds characterised by a chromanol ring with a phytol side chain referred to as tocopherols and tocotrienols. Tocopherols possess a saturated phytol side chain whereas the side chain of tocotrienols have three unsaturated residues. Isomers of these compounds are distinguished by the number and arrangement of methyl substituents attached to the chromanol ring. The predominant isomer found in the body is alpha-tocopherol, which has three methyl groups in addition to the hydroxyl group attached to the benzene ring. The diet of animals is comprised of different proportions of tocopherol isomers and specific alpha-tocopherol-binding proteins are responsible for retention of this isomer in the cells and tissues of the body. Because of the lipophilic properties of the vitamin it partitions into lipid storage organelles and cell membranes. It is, therefore, widely distributed in throughout the body. Subcellular distribution of alpha-tocopherol is not uniform with lysosomes being particularly enriched in the vitamin compared to other subcellular membranes. Vitamin E is believed to be involved in a variety of physiological and biochemical functions. The molecular mechanism of these functions is believed to be mediated by either the antioxidant action of the vitamin or by its action as a membrane stabiliser. alpha-Tocopherol is an efficient scavenger of lipid peroxyl radicals and, hence, it is able to break peroxyl chain propagation reactions. The unpaired electron of the tocopheroxyl radical thus formed tends to be delocalised rendering the radical more stable. The radical form may be converted back to alpha-tocopherol in redox cycle reactions involving coenzyme Q. The regeneration of alpha-tocopherol from its tocopheroxyloxyl radical greatly enhances the turnover efficiency of alpha-tocopherol in its role as a lipid antioxidant. Vitamin E forms complexes with the lysophospholipids and free fatty acids liberated by the action of membrane lipid hydrolysis. Both these products form 1:1 stoichiometric complexes with vitamin E and as a consequence the overall balance of hydrophobic:hydrophillic affinity within the membrane is restored. In this way, vitamin E is thought to negate the detergent-like properties of the hydrolytic products that would otherwise disrupt membrane stability. The location and arrangement of vitamin E in biological membranes is presently unknown. There is, however, a considerable body of information available from studies of model membrane systems consisting of phospholipids dispersed in aqueous systems. From such studies using a variety of biophysical methods, it has been shown that alpha-tocopherol intercalates into phospholipid bilayers with the long axis of the molecule oriented parallel to the lipid hydrocarbon chains. The molecule is able to rotate about its long axis and diffuse laterally within fluid lipid bilayers. The vitamin does not distribute randomly throughout phospholipid bilayers but forms complexes of defined stoichiometry which coexist with bilayers of pure phospholipid. alpha-Tocopherol preferentially forms complexes with phosphatidylethanolamines rather than phosphatidylcholines, and such complexes more readily form nonlamellar structures. The fact that alpha-tocopherol does not distribute randomly throughout bilayers of phospholipid and tends to form nonbilayer complexes with phosphatidylethanolamines would be expected to reduce the efficiency of the vitamin in its action as a lipid antioxidant and to destabilise rather than stabilise membranes. The apparent disparity between putative functions of vitamin E in biological membranes and the behaviour in model membranes will need to be reconciled.     

Membrane Lipids: What Membrane Physical Properties are Conserve during Physiochemically-Induced Membrane Restructuring?

SYNOPSIS. Current theories assert that organisms finely adjustthe order, or fluidity, of their cellular membranes in responseto changes in their physiochemical environment (e.g., pressure,temperature, salinity, etc.). However, membrane order may notbe the only property that is conserved. The most commonly observedalterations in cell membrane composition under conditions ofaltered physiochemical environment, namely changes in the phosphatidylethanolamine/phosphatidylcholine(PE/PC) ratio and the content of highly unsaturated acyl chains,are difficult to fully reconcile with the conservation of membraneorder alone. This report reviews the literature concerning twoproperties of membranes that may play vital roles in the adaptationof cellular membranes to changing environments: a) the tendencyof membranes to relax into the reversed hexagonal phase andb) the occurrence and structure of lipid-driven domains withinthe membrane. The tendency of a membrane to form the reversedhexagonal phase is a property central to a variety of importantcellular events. This tendency is tightly regulated by variationof the ratio of hexagonal phase-forming lipids to lamellar phase-forminglipids in the membrane. In most animal cells, this correspondsto the PE/PC ratio. Highly unsaturated acyl chains, in conjunctionwith cholesterol, modulate the occurrence and structure of lipid-drivenmembrane domains. These membrane domains are also criticallyinvolved in a number of key cellular processes. The changesin membrane lipid composition that occur during adaptation tothe environment may be required for the preservation of thetendency to form nonlamellar phases and of the occurrence andspecific structure of domains within the membrane, in additionto overall membrane order.     

Biosynthesis of phosphatidylethanolamines and phosphatidylcholines from ethanolamine and choline in rat liver.

1. The kinetics of phosphatidylcholine and phosphatidylethanolamine synthesis in rat liver were followed 5-60 min after the intraportal injection of [14-C]choline and [3-H]-ethanolamine. 2. At all time-intervals the specific radioactivity of CDP-choline was only about half that of phosphorylcholine. This indicated that CDP-choline was formed at a similar rate from phosphorylcholine and phosphatidylcholines, the latter probably through the reverse reaction of cholinephosphotransferase (EC 2.7.8.2.). In view of recent data obtained from experiments in vitro this implies a significant role for the cholinephosphotransferase reaction in the turnover of molecular species of phosphatidylcholine. 3. The specific radioactivity of CDP-ethanolamine was about twice that of phosphorylethanolamine at all time-intervals studied. This supports a previous suggestion that the liver phosphorylethanolamine pool is subject to compartmentation and shows that there is no rapid equilibration between different pools. In contrast with a recent study, no evidence was found for any significant methylation of phosphoryl-or CDP-ethanolamine to the corresponding choline derivative. 4. Quantitative data on the biosynthesis of molecular species of phosphoLIPIDS via CDP derivatives were calculated according to simple kinetic models. They were in the same range as those calculated from earlier data on precusors incorporated via diacylglycerols. 5. The proportion of radioactive phosphatidylethanolamines appearing in the plasma was approximately ten times lower than that for phosphatidylcholines. No selectivity was observed in the transfer into plasma of different molecular species of phosphatidylethanolamine.