Biomedical subjects

L Freysz

Publications and source records attributed to L Freysz.

At least 19 recordsLinked to original sources

PubMed1992

Reversibility of the reactions catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase solubilized from rat-brain microsomes.

The incorporation of CMP into CDP-ethanolamine and CDP-choline, catalyzed by ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2), respectively, has been studied in solubilized preparations of rat-brain microsomes. Mn2+ ions were required for the maximal activity of both enzymes. The CMP concentration needed to reach the half-maximal reaction rate was 1.6 microM for both activities. The rate of incorporation of CMP into CDP-choline and CDP-ethanolamine was increased by increasing the concentration of phosphatidylcholine and phosphatidylethanolamine, respectively, in detergent-phospholipid micellar systems. The rate of the reaction at pH 6.5 was comparable with that measured at pH 8.5, whereas the rate of synthesis of phosphatidylcholine and phosphatidylethanolamine, catalyzed by the same enzymes, increased with pH. Ethanolaminephosphotransferase, which catalyzes the synthesis of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerol, was co-eluted with the enzyme activity catalyzing the reverse reaction, when solubilized microsomes were submitted to anion exchange chromatography on DEAE Bio-Gel A. Cholinephosphotransferase was inactivated during the chromatographic procedure.

Animals↗

PubMed1992

Exogenous gangliosides modulate calcium fluxes in cultured neuronal cells.

Previous work from this and other laboratories has shown that the neuritogenic effect due to exogenous gangliosides on primary neurons in culture is accompanied by several morphological and biochemical modifications. The present results indicate that the treatment of these neurons with gangliosides, under the experimental conditions which are known to produce a sprouting effect, inhibited the influx of 45Ca2+ and increased the release of 45Ca2+ from the cells. No significant differences were noted using concentrations of gangliosides (10(-8)-10(-5) M) either below or above the critical micellar concentrations. No apparent specificity was observed among various species of individual sialocompounds (GM1, GD1a). Moreover the presence or absence of fetal calf serum in the culture medium influenced the levels of 45Ca2+ fluxes. This study confirms the hypothesis that gangliosides may be considered as Ca2+ flux modulators in neuronal cells.

Animals↗

PubMed1991

Stimulation of mono- and diacylglycerol lipase activities by gangliosides in chicken neuronal cultures.

Chicken neurons in culture display measurable activities of mono- and diacylglycerol lipases. Treatment of chicken neuronal cultures with gangliosides (10(-8)M to 10(-5)M) resulted in a time and dose dependent increase in monoacylglycerol lipase activity. The diacylglycerol lipase showed significant increase in specific activity before that of monoacylglycerol lipase. The increase was observed only up to 24 hours and no differences between diacylglycerol lipase activity of control and ganglioside treated cells were observed after 48 hours. The data indicate that the treatment of neurons with exogenous gangliosides affect the diglyceride metabolism in stimulating not only the enzymes catalyzing their production but also those involved in their catabolism.

Animals↗

PubMed1991

Effect of monomethylethanolamine, dimethylethanolamine, gangliosides, isoproterenol, and 2-hydroxyethylhydrazine on the conversion of ethanolamine to methylated products by cultured chick brain neurons.

The sequential methylation of ethanolamine and its phosphorylated derivatives has been studied with chick neurons in culture in the presence of several pharmacological agents. Incubation with [3H]ethanolamine in the presence of monomethylethanolamine and dimethylethanolamine indicated that in these neurons the preferential conversion to choline-containing compounds is via the methylation of phosphorylethanolamine. The possibility that there are two separate enzymes, i.e., one responsible for the methylation of water-soluble ethanolamine-containing compounds and another for the ethanolamine phospholipids, was examined with agents believed to influence these conversions. Incubation of neurons in the presence of a mixture of exogenous gangliosides at 10(-8) M and 10(-5) M concentrations showed that these neuritogenic compounds stimulate the methylation of phosphatidylethanolamine and decrease that of phosphorylethanolamine. The inhibitor of phosphatidylethanolamine methyltransferase (EC 2.1.1.17), 2-hydroxyethylhydrazine, decreased the conversion of phosphatidylethanolamine to phosphatidylcholine and increased that of phosphorylethanolamine to phosphorylcholine. The possible effects of adrenergic stimulation were studied by the incubation of neurons with isoproterenol at 10(-6) M and 10(-5) M concentrations. There was a reduction of phosphorylethanolamine methylation and a stimulation of that of phosphatidylethanolamine, and these effects were counteracted by the presence of 5 x 10(-5) M propranolol.

Animals↗

PubMed1990

Sensitive analysis of phospholipid molecular species by high-performance liquid chromatography using fluorescent naproxen derivatives of diacylglycerols.

A sensitive high-performance liquid chromatographic (HPLC) method for the separation and determination of diacylglycerophospholipid and diacylglycerol (DAG) molecular species has been developed. Phospholipids are hydrolysed with phospholipase C and the resulting DAGs are reacted with naproxen chloride in the presence of 4-dimethylaminopyridine. The naproxen-DAGs were purified by thin-layer chromatography on silica gel G plates. Molecular species were separated using reversed-phase HPLC with isocratic elution and determined by measuring the absorbance at 230 nm or fluorescence at 352 nm (excitation at 332 nm). The method was applied to the determination of diacylglycerophosphoethanolamine in rat cerebrum and cerebellum. The molar absorption coefficient of the naproxen derivatives was 53,000 lmol-1 cm-1 at 230 nm, permitting the generation of linear concentration-dependent determinations down to less than 10 pmol. A ten-fold increase in sensitivity was obtained with a fluorescence detection system owing to the fluorescent properties of the proposed adduct.

Animals↗

PubMed1990

A rat brain cytosolic N-methyltransferase(s) activity converting phosphorylethanolamine into phosphorylcholine.

It had been previously speculated upon but never proved that the methylation of phosphorylethanolamine could contribute to the production of choline containing compounds. However, experimental evidence obtained with neuronal cultures was interpreted as showing that the stepwise methylation of phosphobases may be an important route for this biosynthesis. We demonstrate that cytosolic fraction from rat brain possesses a N-methyltransferase activity capable of methylating phosphorylethanolamine and its mono- and dimethyl-derivatives into phosphorylcholine. The level of activity detectable in rat liver cytosol is only 18% of that found in the brain cytosol.

Animals↗

PubMed1990

In vitro synthesis and transbilayer movement of phosphatidylethanolamine molecules labelled with different fatty acids in chick brain microsomes.

The transbilayer fatty acid distribution of diacylglycerophosphoethanolamine and the translocation of newly synthesized phosphatidylethanolamine molecules labelled with different fatty acids has been investigated in chick brain microsomes using trinitrobenzensulfonic acid. The determination of the fatty acid composition of diacylglycerophosphoethanolamine in both the outer and the inner leaflet of the microsomal vesicles revealed a similar distribution indicating that both leaflets share the same molecular species. The in vitro incorporation of radioactive fatty acids (16:0, 18:1 and 20:4(n-6] into ethanolamine phospholipids, known to be catalyzed by the lyosphosphatidylethanolamine acyl transferase, showed that the radioactive diacylglycerophosphoethanolamine molecules appeared first in the outer leaflet and were thereafter transferred to the inner leaflet. The apparent rate of translocation of the newly synthesized ethanolamine phospholipid molecules was the highest for those labelled with 16:0 and the lowest for those labelled with 20:4(n-6). The results indicate that the active site of the acyl-CoA:lysophosphatidylethanolamine acyltransferases is located on the outer leaflet of the microsomal vesicles and that the different newly synthesized molecular species of diacylglycerophosphoethanolamine may be translocated from the outer to the inner leaflet at different rates.

Animals↗

PubMed1990

Molecular species of choline and ethanolamine phospholipids in rat cerebellum during development.

The molecular species composition of rat cerebellar phospholipid subclasses has been studied by HPLC after phospholipase C treatment and dinitrophenyl derivatization. During rat cerebellum development (3-90 days postpartum), cholinephosphoglycerides and ethanolamine phosphoglycerides represented approximately 80% of all phospholipids, with their relative amount changing after 1 month. Among ethanolamine phosphoglycerides, the molar ratio of diacylglycerophosphoethanolamine (diacylGPE) to alkenylacylGPE decreased from approximately 1.4 at 3 days to approximately 0.5 after 10 days. The phospholipids investigated contained up to 12 different molecular species. The rate of accumulation of the various molecular species of diacylglycerophosphocholine (diacylGPC), diacylGPE, and alkenylacylGPE during cerebellar development allowed a classification into three main groups. The overall increase of the molecular species of the first group (6-diacylGPC, 5-diacylGPE, and 4-alkenylacylGPE) was approximately 18-fold between 3 and 90 days, with a faster rate of accumulation between 3 and 30 days. Those of the second group (3-diacylGPC, 5-diacylGPE, and 5-akenylacylGPE) increased by approximately 45-fold during the same developmental period, at a slow rate before day 15 and a faster one thereafter. The molecular species of the third group (3-alkenylacylGPE) increased by greater than 250-fold between 3 and 90 days, at a very slow rate before day 21 and more quickly thereafter. The different rates of accumulation of the components of the three groups during cerebellar development suggest a preferential location of the first group in membranes of neuronal perikaryons, glial cells, and synaptosomal structures. Those of the second group appear to be located in both synaptosomal membranes and myelin sheets, and those of the third group can be considered as myelin markers.

Animals↗

PubMed1989

Conversion of ethanolamine, monomethylethanolamine and dimethylethanolamine to choline-containing compounds by neurons in culture and by the rat brain.

The incubation of neurons from chick embryos in primary culture with [3H]ethanolamine revealed the conversion of this base into monomethyl, dimethyl and choline derivatives, including the corresponding free bases. Labelling with [methyl-3H]monomethylethanolamine and [methyl-3H]dimethylethanolamine supported the conclusion that in chick neuron cultures, phosphoethanolamine appears to be the preferential substrate for methylation, rather than ethanolamine or phosphatidylethanolamine. The methylation of the latter two compounds, in particular that of phosphatidylethanolamine, was seemingly stopped at the level of their monomethyl derivatives. Fetal rat neurons in primary culture incubated with [3H]ethanolamine showed similar results to those observed with chick neurones. However, phosphoethanolamine and phosphatidylethanolamine and, to a lesser extent, free ethanolamine, appeared to be possible substrates for methylation reactions. The methylation of water-soluble ethanolamine compounds de novo was further confirmed by experiments performed in vivo by intraventricular injection of [3H]ethanolamine. Phosphocholine and the monomethyl and dimethyl derivatives of ethanolamine were detected in the brain 15 min after injection.

Animals↗

PubMed1989

Effect of pyridoxal 5'-phosphate and valproic acid on phospholipid synthesis in neuroblastoma NA.

Phospholipid metabolism in neuroblastoma cells in monolayer culture after acute exposure to pyridoxal phosphate (PLP) has been studied. (a) A strong depression of the rate of biosynthesis of cellular phospholipids from labeled choline and ethanolamine, is demonstrated in neuroblastoma cells grown in culture media containing PLP. (b) Valproic acid reverses the effect of PLP on ethanolamine and choline incorporation into cell lipid. Other anticonvulsants (clonazepam, diazepam, carbamazepine, diphenylhydantoin and ethosuximide) have little or no effect on reversing the inhibition of lipid synthesis produced by PLP. (c) PLP decreases the cellular uptake of choline. This effect might be responsible for the decreased lipid synthesis and is partially reversed by valproic acid. (d) The energy charge of the cell is not affected by either PLP or valproic acid, but it is diminished by the two compounds together. (e) The degradation of choline lipids is decreased by PLP and valproic acid. The hydrolysis of phosphocholine and the outflow of choline from cultured cells is also affected by the drugs. Variations of ethanolamine and choline transport should not be due to any effects of PLP or valproic acid on the lipid phase of the membranes since these molecules have no effect on the permeability of liposomes. (f) It is concluded that ethanolamine and choline lipid metabolism in cultured neuroblastoma cells is influenced by PLP and/or valproic acid, probably through a mechanism involving the transport of precursors across the membrane, although other mechanisms cannot be ruled out.

Energy Metabolism↗

PubMed1989

An improved procedure for the purification of ethanolaminephosphotransferase. Reconstitution of the purified enzyme with lipids.

Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) has been purified in active form from rat brain microsomes by a two-step chromatographic procedure. Enzyme preparations characterized by high specific activity and stability were obtained supplementing the solubilization and elution buffers, containing 1% Triton X-100, with 0.01% 2,6-di-tert-butyl-4-methylphenol. The specific activity of the purified enzyme was about 1200-times higher than that of the crude solubilized enzyme. The lipid dependence of ethanolaminephosphotransferase was studied both in the presence of Triton X-100 and in detergent-free enzyme preparations. The activity of the detergent-solubilized ethanolaminephosphotransferase was strongly modified by phospholipids. The kinetic behaviour of the enzyme was also dependent on the lipids contained in the aggregates obtained by removal of the detergent from detergent/lipid/protein suspensions. A regulatory role of phospholipids on the activity of the membrane-bound ethanolaminephosphotransferase is discussed.

Animals↗

PubMed1989

Choline plasmalogen synthesis by the methylation pathway in chick neurons in culture.

Choline plasmalogens represent a minor component of lipid membranes in most tissues. In spite of this, their rapid turnover indicates a possible functional role in the cell. The present study demonstrates that these compounds can be synthesized in neuronal cell cultures from chick embryo hemispheres by methylation of ethanolamine plasmalogens since choline plasmalogens were labeled after incubation of cells with tritiated ethanolamine or methionine. This finding could be of a particular interest since it has been suggested that choline plasmalogens, synthesized by methylation, might be involved in receptor activation.

Animals↗

PubMed1988

Effect of exogenous gangliosides on the lipid composition of chick neurons in culture.

When exogenous gangliosides are added to the growth medium of neuronal cell cultures they are inserted into their plasma membranes and are afterwards metabolized in the cytoplasmic interior. The action of exogenous gangliosides brings important morphological and biochemical changes to neurons in culture. The present report shows that the treatment with exogenous gangliosides of a primary culture of chick neurons modified the distribution of fatty acids in phosphatidylinositol (PI), mainly that of arachidonic acid and the fatty acids of the (n - 3) series without affecting the other phospholipids. The composition of neutral lipids did not change but their content was increased up to 2-3-fold depending upon the concentration of gangliosides. The change of the growth medium from one containing fetal calf serum to a chemically defined one reduced dramatically the content of free fatty acids while the addition of gangliosides raised this content to normal levels. The increase in the amount of diacylglycerol (DG) confirmed the finding that gangliosides stimulate phosphoinositide degradation. Finally the fatty acid composition of DG suggests indirectly that this compound might be produced also by degradation of phosphatidylcholine and not only of PI.

Animals↗

PubMed1988

Topological distribution of choline phospholipid fatty acids in trout intestinal brush-border membrane.

The transbilayer distribution of choline phospholipids in trout intestinal brush-border membrane has been investigated using phospholipase C (from Clostridium welchii). In the middle intestine, 84% of phosphatidylcholine (PC) and 60% of sphingomyelin (SP) are located in the outer membrane leaflet. In the posterior intestine, 89% of PC and 52% of SP are located in the outer membrane leaflet. The externally located PC molecular species are (n - 3) fatty acid-rich in both parts of the intestine. While the sphingomyelin molecular species containing 24:1(n - 9) are exclusively located in the outer leaflet in the middle intestine, those containing 14:0 are more abundant in the same leaflet but in the posterior intestine. This strongly asymmetric distribution of both choline phospholipids may have numerous consequences on the brush-border membrane characteristics.

Animals↗

PubMed1987

Topological distribution of aminophospholipid fatty acids in trout intestinal brush-border membrane.

The transbilayer distribution of aminophospholipids in trout intestinal brush-border membrane has been investigated using trinitrobenzene sulfonic acid (TNBS). In the middle intestine, phosphatidylethanolamine (PE) is symmetrically distributed between the two leaflets while 68% of the phosphatidylserine (PS) are located in the inner membrane leaflet. In the posterior intestine, 64% of the PE and 69% of the PS are located in the inner membrane leaflet. When asymmetrically distributed, the inner species of PE and PS have a higher content of 22:6(n-3) than the outer ones. This asymmetric distribution of docosahexaenoic acid in trout intestinal brush-border membrane might be related to the rod-like shape of the microvillus membrane and to its metabolism to hydroxylated derivatives.

Animals↗

PubMed1987

Partial purification of ethanolaminephosphotransferase from rat brain microsomes.

Rat brain ethanolaminephosphotransferase (CDPethanolamine : 1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) was solubilized by treating rat brain microsomes with buffered solutions containing octyl glucoside or Triton X-100. The solubilized enzyme was stable both at 4 degrees C and at -18 degrees C. A partial purification was obtained using an ion-exchange chromatographic procedure. The partially purified enzyme showed four major bands in SDS-polyacrylamide gel electrophoresis; its specific activity was increased by a factor of 37 compared to that of the membrane-bound enzyme. Glycerol and diacylglycerol were effective as stabilizers. Phosphatidylcholine, lysophosphatidylcholine and phosphatidylserine increased both the specific activity and the stability of the partially purified enzyme.

Animals↗

PubMed1987

Ethanolamine base exchange in astrocyte primary cultures: localization and developmental studies.

The enzymatic activities of ethanolamine base exchange (EBEE) and CDP-ethanolamine: 1,2-diacylglycerol ethanolamine phosphotransferase (EPT) were investigated during the growth of rat astrocyte primary cultures. From the 16th day, cells ceased to divide (2.0 X 10(6) cells per culture dish); the total phospholipid (PL) content increased 1.5 fold between the 16th and 24th day (0.20 to 0.30 mumol per mg protein) but the amount of ethanolamine phospholipid (28% of PL content) remained constant. Whereas the specific activity (pmol/min X mg protein) of EPT reached a plateau at 16 days in culture and remained constant (400) thereafter, that of EBEE increased up to the 19th day (190) and decreased gradually to a basal level (75) at the 24th day. EBEE activity was not detected in plasma membranes isolated from 16, 19 and 24 days astrocyte cultures. Sub-cellular fractionation and determination of EBEE specific activities showed that the 104 X 10(3) g fraction (P4) was 4.8 and 8.8 fold enriched at the 16th day and 24th day respectively as compared to the whole cell homogenate (50 and 75). The 7 X 10(3) g (P2) and 17 X 10(3) g (P3) fractions were 8.4 and 7.0 fold enriched respectively at the 19 day in culture. The percentages of the enzymatic activity in the different subcellular fractions were 30, 57.2 and 25.7 for P2 and 39.2, 2.6 and 39.8 for P4 at 16, 19 and 24 days in culture respectively. The activity remained constant in P3 (23%) and was negligible in P1 (6%). Ultrastructural studies revealed that P2 and P3 were enriched in mitochondria while P4 contained essentially microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Phospholipid fatty acyl group composition in mitochondria, microsomes and plasma membranes isolated from rat astrocyte primary cultures: developmental studies.

The fatty acid composition of choline, ethanolamine and serine phospholipids (PC, PE and PS) in heavy mitochondria, light mitochondria, microsomes and plasma membranes isolated from 16-, 19- and 24-day-old cultured rat astrocytes was investigated; within this period cultures reached confluency and ceased to divide. Plasma membranes revealed a stable distribution of the major fatty acids (FA) present in PC, PE and PS. The FA pattern of PC was similar to that of PE and PS. Analysis of the FA composition of PC, PE and PS found in intracellular membranes indicates that each major FA displayed a typical variation depending on the organelle and the phospholipid studied. Schematically for both phospholipids, PC and PE, the palmitic acid content increased in most subcellular fractions between 16 and 24 days; this variation was compensated by the decrease of stearic acid. Arachidonic acid level was very low at 16 days and increased markedly up to 24 days; thus a decrease of the saturated to unsaturated FA ratio (delta:0/delta:1, 2, 4) occurred. In contrast PS, which was poor in arachidonic acid between 16 and 24 days, gave rise to delta:0/delta:1, 2, 4. These results suggest (1) a reorganisation of the phospholipid hydrophobic core in mitochondria and endoplasmic reticulum when density-dependent inhibition phenomena occurs, and (2) a 'buffering' mechanism which protects the plasma membrane from marked changes of its phospholipid FA composition.

Animals↗