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PubMed1984

Phospholipid content and composition of human meningiomas.

The content and distribution of phospholipids, as well as their fatty acid composition, were studied in 16 human meningiomas in comparison with normal leptomeninges. The total phospholipid content of tumors (expressed as organic phosphorus/mg DNA) was similar to that of the tissue from which they originated. The same phospholipid classes were present in both tissues, but with a different pattern: an increase of phosphatidylinositol and phosphatidylcholine and a decrease of sphingomyelin and alkenyl-acyl phosphatidylethanolamine were detected in meningiomas. Stearic, palmitic, and oleic acids were the major phospholipid fatty acids in both leptomeninges and meningiomas. However, remarkable differences between the fatty acid composition of the two tissues were shown. The most striking difference is a marked increase of unsaturated fatty acids in tumors associated with a decrease of saturated forms. This feature is common to all major phospholipid classes. The possible significance and role of phospholipid modifications in the functional properties of meningioma plasma membrane is discussed.

Adult↗
PubMed1999

Negatively charged phospholipids influence the activity of the sensor kinase KdpD of Escherichia coli.

Synthesis of the high-affinity K(+)-translocating Kdp-ATPase of Escherichia coli, encoded by the kdpFABC operon, is regulated by the membrane-bound sensor kinase KdpD and the soluble response regulator KdpE. K(+) limitation or a sudden increase in osmolarity induces the expression of kdpFABC. Due to the importance of K(+) to maintain turgor, it has been proposed that KdpD is a turgor sensor. Although the primary stimulus that KdpD senses is unknown, alterations in membrane strain or the interaction between KdpD and membrane components might be good candidates. Here, we report a study of the influence of the membrane phospholipid composition on the function of KdpD in vivo and in vitro using various E. coli mutants defective in phospholipid biosynthesis. Surprisingly, neither the lack of the major E. coli phospholipid phosphatidylethanolamine nor the drastic reduction of the phosphatidylglycerol/cardiolipin content influenced induction of kdpFABC expression significantly. However, in vitro reconstitution experiments with synthetic phospholipids clearly demonstrated that KdpD kinase activity is dependent on negatively charged phospholipids, whereas the structure of the phospholipids plays a minor role. These results indicate that electrostatic interactions are important for the activity of KdpD.

Adenosine Triphosphatases↗
PubMed2005

Chronic valproate does not alter the kinetics of docosahexaenoic acid within brain phospholipids of the unanesthetized rat.

RATIONALE: It has been reported that each of three drugs effective in treating bipolar disorder (lithium, carbamazepine, and valproate) decreases the turnover of arachidonic acid (AA, 20:4n-6) in brain phospholipids of the awake rat. It is also known that lithium and carbamazepine do so without decreasing the turnover of docosahexaenoic acid (DHA, 22:6n-3). OBJECTIVE: The aim of this study was to see whether valproate also specifically targets the turnover of AA but not of DHA in brain phospholipids. METHODS: Valproate was administered (200 mg kg(-1), i.p.) to rats for 30 days to produce a therapeutically relevant plasma concentration and then determine its effect compared with that of vehicle on incorporation and turnover rates of DHA in brain phospholipids. In unanesthetized rats that had received valproate or vehicle, [1-14C]DHA was infused intravenously, and arterial blood plasma was sampled until the animal was killed at 5 min; and its brain, after being microwaved, was subjected to chemical and radiotracer analysis. RESULTS: Using equations derived from our fatty acid model, it was found that chronic valproate compared with vehicle did not alter the rate of incorporation or turnover of DHA in brain phospholipids. Valproate-treated animals had higher concentrations of linoleic acid (18:2n-6) in several brain phospholipids, supporting the hypothesis that it alters brain n-6 fatty acid metabolism. CONCLUSIONS: The results, comparable to published findings following chronic administration of lithium and carbamazepine to rats, support the hypothesis that drugs are effective against mania in bipolar disorder act by downregulating incorporation and turnover of AA, but not of DHA, in brain phospholipids.

Acyl Coenzyme A↗
PubMed1999

Temperature dependence of membrane lipid composition in early blastula embryos of Lytechinus pictus: selective sorting of phospholipids into nascent plasma membranes.

Lytechinus pictus eggs were fertilized and incubated at 10, 16, and 23 degrees C until the early blastula stage of embryonic development. The phospholipid composition of the embryos and control unfertilized eggs remain identical and unchanged as incubating temperatures are varied; thus, neither incubating temperature, fertilization nor membrane assembly affect their total phospholipid composition. This result agrees with metabolic studies by others, using only a single incubation temperature, and indicates that embryonic development to the early blastula stage occurs with little, if any, de novo phospholipid biosynthesis. However, as in all poikilotherms, the phospholipid composition of the nascent plasma membranes varies with the incubation temperature. Thus, until the blastula stage of embryonic development, the lipids of these newly formed plasma membranes are derived from lipid pools within the embryo whose phospholipid composition is static. The variation of plasma membrane composition is primarily reflected in an increase in the phosphatidylethanolamine (PE): phosphatidylcholine (PC) ratio as incubating temperatures decrease; this is achieved by an exchange of PE for PC. Several mechanisms are considered for the specificity of the selective sorting and assembly of these phospholipids into the nascent plasma membranes.

Animals↗
PubMed2000

Phospholipid compositional changes of five pseudomonad archetypes grown with and without toluene.

Bacterial physiological responses to toluene exposure were investigated in five reference pseudomonad strains that express different toluene degradation pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1. The intact phospholipids of these archetypes, grown with and without toluene, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. All strains showed significant changes in phospholipid content and composition as an adaptive response to toluene exposure, as well as considerable diversity in response mechanisms. For example, the phospholipid content of toluene-grown PKO1, F1, and KR1 were 10.9-34.7% of that found in succinate-grown strains, while the phospholipid content of mt-2 and G4 increased by 56% and 94%, respectively, when grown on toluene. In addition, PKO1, F1, and mt-2 responded to the presence of toluene by synthesizing more phosphatidylglycerol, whereas G4 and KR1 synthesized phospholipids with polyunsaturated fatty acids (C18:2) on one or both of the sn-2 positions. These changes in phospholipid composition and concentration probably reflect the sensitivity and degree of tolerance of these strains to toluene, and suggest that different mechanisms are utilized by dissimilar bacteria to maintain optimal lipid ordering in the presence of such environmental pollutants.

Burkholderia↗
PubMed2006

The inhibition of tumor cell adhesion on human mesothelial cells (HOMC) by phospholipids in vitro.

BACKGROUND AND AIMS: Intraperitoneal tumor cell adhesion to extracellular matrix and to mesothelial cells mediated by integrins is an important step in developing peritoneal carcinosis. In former animal studies, we could demonstrate that intraperitoneal treatment with a new phospholipid (PL) emulsion significantly reduces the amount of peritoneal carcinosis by adhesion prevention. This in vitro study tries to elucidate the influence of phospholipids on cells of the human gastric cancer cell line (NUGC-4) and the human rectal cancer cell line (HRT-18) adhering to mesothelial cells (HOMC) in a monolayer culture in vitro. MATERIALS AND METHODS: HOMC cells were derived from omentum majus from patients undergoing elective abdominal surgery. Three passages of both cancer cell lines (NUGC-4 and HRT-18) were used. 1x10(5)/100 microl (HRT-18) or 1.2x10(5)/100 microl (NUGC-4) cells, according to forgoing dilution series, were pretreated with different concentrations of phospholipid emulsion (0.05, 0.1, 0.5, 0.75, 1% PL) stained with cell tracker chloromethyl-benzamidodialkylcarbocyanine (CM-DIL) and seeded into each well on the mesothelial monolayer. After 90 min, the number of adherent cells was counted by fluorescence microscopy at 530 and 620 nm. Additionally, flow cytometric analysis of integrin alpha3 and beta1 expression on the tumor cell surface after treatment with phospholipids was completed. RESULTS: We found a dose dependent effect of phospholipids on both tumor cell lines causing a reduction of cell-cell adhesion. Already low concentrations of phospholipids (PL 0.5) had a significant influence. The mean cell count could be reduced from 234+/-12/mm2 in controls to 124+/-41/mm2 (PL 0.5; NUG-4) and from 295+/-49/mm2 to 169+/-29/mm2 (PL 0.5; HRT-18), respectively. Additionally, the integrin alpha3 and beta1 expression on both cell lines could be reduced. CONCLUSION: Our results within the scope of published data indicate that adhesion prevention is capable to reduce peritoneal carcinosis.

Anticarcinogenic Agents↗
PubMed1999

Maternal plasma homocysteine, placenta status and docosahexaenoic acid concentration in erythrocyte phospholipids of the newborn.

UNLABELLED: The enhanced transport of long-chain polyunsaturated fatty acids, in particular docosahexaenoic acid (22:6 omega-3) (DHA), to the fetus is a placental function important for adequate membrane phospholipid formation and herewith decisive for the quality of fetal CNS myelination. A compromised placental function is correlated with signs of vascular pathology. As elevated plasma total homocysteine (tHcy) concentrations are considered an independent risk for premature occlusive vascular disease, the influence of maternal plasma tHcy concentrations on placental function was indirectly studied, determining the DHA content in erythrocyte membrane phospholipids of the newborn. A total of 60 unselected pregnant women (age range: 21 to 39 years) were investigated at delivery. Gestational age ranged from 26 to 41 weeks. Prior to delivery a placental ultrasound scan was performed. Complete sets of data could be obtained from 43 mothers and their offspring. tHcy concentrations were determined in the plasma of cord and maternal blood. The fatty acid pattern of erythrocyte membrane phospholipids was determined in the mothers and their newborns. Z-scores of the birth weights ranged from -3.4 to 2.1 and of the placental weights from -3.8 to 4.7. The mean maternal plasma tHcy concentration was 6.29 +/- 3.34 micromol/l ranging from below our limit of detection up to 15 micromol/l. These maternal concentrations were correlated with those of their infants (r = 0.71; P < 0.0001). The tHcy concentrations were significantly higher in mothers with pregnancies complicated by gestosis or placental calcifications. The Z-scores of birth weights as well as placental weights showed a significant negative correlation with maternal plasma tHcy concentrations. The mean DHA percentage of total fatty acids in erythrocyte phospholipids was 3.2 +/- 2.2% in the mothers and 3.4 +/- 2.3% in their newborns. Most importantly the maternal plasma tHcy levels and the erythrocyte phospholipid DHA concentrations of their offspring were significantly correlated (r = -0.51; P < 0.0003). CONCLUSION: In this study, total homocysteine concentrations were elevated in the plasma of pregnant women with signs of placental vasculopathy. Maternal plasma total homocysteine concentrations were positively correlated with erythrocyte phospholipid docosahexaenoic acid of their offspring and may be an indicator for the integrity of placental vascular function. The nutritional status as well as predisposing genetic factors of pregnant mothers need to be investigated more thoroughly.

Adult↗
PubMed2000

Phospholipid identification and quantification of membrane vesicle subfractions by 31P-1H two-dimensional nuclear magnetic resonance.

An approach to the direct quantification of phospholipids from two-dimensional 31P-1H nuclear magnetic resonance (NMR) spectroscopy with isotropic proton mixing has been developed as a general method for phospholipid analysis of minor membrane vesicle subfractions. Membrane vesicles were subfractionated by sedimentation to density equilibrium in a sucrose gradient, and a modified Folch method was employed to extract their phospholipids. The coefficient for the NMR detection efficiency of each phospholipid and the relative mole percentage of the phospholipids present in the membrane vesicles were calculated. We demonstrate low detection limits such that relative concentrations of phospholipids in membrane subfractions may be determined even in the submicromolar range.

Animals↗
PubMed2000

Utilization of docosahexaenoic acid from intravenous egg yolk phospholipid.

Docosahexaenoic acid (DHA, 22:6n-3) is provided directly to human premature infants during parenteral nutrition from the egg yolk fraction of an intravenous fat emulsion. This study aimed to determine whether the high egg yolk phospholipid content of Intralipid 10% (IL 10%, Pharmacia, Uppsala, Sweden) relative to the standard emulsion Intralipid 20% (IL 20%, Pharmacia) could be a strategy to increase the delivery of DHA to the developing brain. Male, Large White piglets were randomly selected from sows 3 d after birth. Piglets were assigned to receive a 9-d continuous intravenous infusion commencing 5 d after birth of either Intralipid (IL) 10%, IL 20%, or Lipofundin S 20% (LFS; B. Braun, Melsungen, Germany). There were four piglets in each treatment group. IL 10% provides twice as much DHA as IL 20%, while LFS provides no DHA. Protein and other nutrients were provided enterally using a low-fat milk formula. After 9 d, animals were killed, and the fatty acid compositions of blood, liver, and cerebral cortex were analyzed. IL 10% infusion approximately doubled the amount of plasma phospholipid DHA (microg/mL of plasma) in comparison to IL 20%. However, red blood cells, liver, and cerebral cortex phospholipid DHA levels were indistinguishable between these two groups. LFS was associated with reduced levels of DHA in plasma, red blood cell and liver phospholipids in comparison to IL 20%. We conclude that infusion of additional phospholipid is an ineffective strategy for increasing DHA delivery to piglet tissues. This may be due to the formation of inert phospholipid particles in plasma. The data do not support the concept of using IL 10% as a means of providing additional DHA to premature human infants.

Animals↗
PubMed1991

An original method for rapid serial determination of phospholipid biosynthesis. Applications to mammalian lymphocytic cells and a lower eucaryote, Plasmodium falciparum.

A rapid, convenient, and efficient method is presented to measure phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol biosynthesis from [3H]choline, [3H]ethanolamine, and [3H]inositol, respectively. After incubation of the cells in 96-multi-well dishes with the appropriate radioactive precursor, cells were lysed with water and the water-insoluble materials, particularly cellular membranes which contain the bulk of phospholipids, were serially collected on glass-fiber papers using a cell harvester. The method was first applied to human lymphocytic cell lines then adapted to Plasmodium falciparum-infected human erythrocytes which in both cases allowed recovery of more than 90% of the newly biosynthesized phospholipids. With this quick method, adapted to short incubation periods (less than 5 h), we were able to determine optimal conditions such as the best medium (RPMI devoid of serum, thus avoiding interference from endogenous precursors, notably choline present in significant quantities in serum) and the lowest specific activity to be used for each radioactive precursor and the minimum quantity of cells. This method could be adapted to other cell systems, provided that the precursors are specific to phospholipids and that the bulk of biosynthesized phospholipids are present as membrane components. Finally, by this method the activity of effectors of phospholipid metabolism can be tested on a large scale, thus allowing rapid screening of original molecules specifically affecting cellular phospholipid metabolism.

Amino Alcohols↗
PubMed1984

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane.

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.

Animals↗
PubMed1988

Phosphatidylglycerol-modulated protein kinase activity from human spleen. II. Interaction with phospholipid vesicles.

Phosphorylation of endogenous and artificial protein substrates by protein kinase P is stimulated by phosphatidylinositol or phosphatidylglycerol (D. J. Klemm, and L. Elias (1987) J. Biol. Chem. 262, 7580-7585; L. Elias and A. Davis (1985) J. Biol. Chem. 260, 7023-7028). Stimulation of protein kinase P activity required phospholipid vesicles rather than free phospholipid molecules. Protein kinase P activity increased as the phosphatidylinositol content of the vesicles was raised from 20 to 100%; no stimulation was detected below 20% phosphatidylinositol. This suggests that a vesicle surface rich in phosphatidylinositol is required for enzyme activation. Maximum activation of protein kinase P activity showed an optimum value with respect to phospholipid concentration, with both endogenous and artificial protein substrates. The phospholipid concentration at which optimal enzyme activity occurred shifted in response to the concentration of protein substrate, but not enzyme concentration. Therefore, the density of substrate molecules on the surface of phospholipid vesicles is a critical feature of protein kinase P stimulation. Binding of protein kinase P to vesicles was independent of micelle composition, but the binding of the artificial substrate, histone H2B, was specific for vesicles containing phosphatidylinositol or phosphatidylglycerol, and increased as the content of phosphatidylinositol was increased. Thus, an important feature of protein kinase P activation appeared to be the specific binding of protein substrate to phospholipid vesicles.

Binding Sites↗
PubMed1990

Peroxidative modification of phospholipids in myocardial membranes.

Rat heart myocardial membranes exposed to the free radical generating system, Fe2+/ascorbate, undergo lipid peroxidation as evidenced by the accumulation of thiobarbituric acid-reactive substances, loss of polyunsaturated fatty acids from phospholipids, and formation of conjugated dienes and fluorescent substances. In addition, the treated membranes exhibit a dramatic decrease in extractable phospholipids. This decrease is even more pronounced in individual phospholipid classes isolated by high-performance liquid chromatography. The decrease in lipid phosphorus under oxidant stress is accompanied by an increase in the phosphorus content of the aqueous phase after Folch extraction and by an even greater increase of phosphorus in the protein residue. In addition, increased amounts of saturated and monounsaturated fatty acyl groups are found in the protein residue of Fe2+/ascorbate-treated membranes. Extraction of the oxidant-treated membranes with acidic solvents does not enhance the recovery of phospholipids and neither does treatment with detergents, trypsin, and chymotrypsin prior to lipid extraction. However, treatment with the bacterial protease, Pronase, markedly enhances the recovery of phospholipids from the peroxidized membranes. These results indicate that membrane phospholipids undergoing free radical-induced peroxidation may form lipid-protein adducts, which renders them inextractable with lipid solvents.

Animals↗
PubMed1978

Preparation and characterization of phospholipid-depleted chloroplasts.

Spinach class II chloroplasts were treated with snake venom phospholipase A2 in the presence of bovine serum albumin, and separated by sucrose-density centrifugation. The treatment yielded phospholipid-depleted chloroplasts which had lost 82.6% of the original phospholipids. About 20% of the phospholipids of chloroplasts were resistant to enzyme attack. These results suggest that phospholipids exist in two states in chloroplast membranes. In spite of considerable phospholipid depletion, the chloroplast preparations retained a large portion of their photoactivities, i.e. light-induced electron transport, light-induced H+ uptake, and light-induced shrinkage. However, cyclic photophosphorylation was significantly affected with the phospholipid removal.

Chloroplasts↗
PubMed1977

Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum.

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidycholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A2 of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphinogomyelin which is not hydrolysed by the former enzyme.

Animals↗
PubMed1978

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells.

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin. Following supplementation with saturated fatty acids of longer than 15 carbons (100 micron) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth. Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35--41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72--82% after addition of unsaturated fatty acids. A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids. The results suggest that maintenance of membrane fluidity by unsaturated fatty acids in membrane phospholipids is critical to membrane integrity and cell growth.

Cell Division↗
PubMed1978

Evidence for isotropic motion of phospholipids in liver microsomal membranes. A 31P NMR study.

1. The motional properties of phospholipids in bovine and rat liver microsomes and aqueous dispersions of the extracted lipids have been investigated employing 31 P NMR techniques. 2. The 31P NMR spectra obtained from the microsomes indicate that a considerable portion of the constituent phospholipids experience isotropic motion on the NMR timescale (10(-5) s). This is in strong contrast to the spectra obtained from aqueous dispersions of the extracted lipids, which display the characteristic lineshape associated with liquid crystalline phospholipids in (large) bilayer structures, which experience restricted anisotropic motion. 3. Evidence is presented which strongly suggests that the isotropic motion of microsomal phospholipids does not arise from tumbling of the microsomal vesicles or from lateral diffusion of phospholipids around these vesicles. 4. These results are discussed in terms of possible transitory formation of intramembrane non-bilayer lipid configurations, with which the bulk (bilayer) phospholipids are in rapid exchange.

Animals↗
PubMed1978

A new assay system of phospholipid exchange activities using concanavalin A in the separation of donor and acceptor liposomes.

A new assay system of phospholipid exchange activities is described. The exchange activities were quantitated by measuring the stimulation of phospholipid transfer between two separate populations of liposomes, which contained, as the major constituents, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and cholesterol in molar ratios of 6 :2 : 1: 1: 5. One population of the liposomes was made reactive to concanavalin A by the incorporation of 1.8 mol% alpha-D-mannosyl-(1 leads to 3)-alpha-D-mannosyl-sn-1, 2-diglyceride from Micrococcus lysodeikticus. The concanavalin A-reactive liposomes, a phospholipid donor, were doubly labelled with [6-3H] galactosylglucosyl ceramide and that class of 32P-labelled phospholipids whose exchange was being measured. The 3H-labelled glycolipid served as a non-exchangeable reference marker. The other population of the liposomes, a phospholipid acceptor, was concanavalin A nonreactive. These two populations of liposomes were incubated with the cytosol protein of rat liver in a total volume of 0.2 ml. After the incubation, two different procedures were used to separate the two liposomal populations. In one procedure concanavalin A was added to agglutinate the reactive liposomes; the flocculated lectin . liposome complex was separated from the non-reactive liposomes by brief centrifugation. In the other procedure the reactive liposomes were trapped by binding to concanavalin A covalently coupled to Sepharose 2B; the complex was separated from the non-reactive liposomes by filtration through a filter paper under suction. In both assay procedures the amount of phospholipid transferred from the donor to the acceptor liposomes was calculated from the decrease of 32P/3H ratio of the concanavalin A-reactive liposomes during the incubation. By the assasy system it is possible to determine phosphatidylcholine and phosphatidylinositol exchange activities in 100 micrograms of rat liver cytosol protein.

Animals↗