Acylation of endogenous acyl acceptors by mouse sciatic nerve microsomes.

Phospholipid (chiefly phosphatidylcholine) labeling from radioactive acyl-CoAs by mouse sciatic nerve microsomes is observed in the absence of added acyl acceptors. The maximal acylation (ca 10% of administered) for 10 micrograms microsomal proteins is observed at relatively low amounts of oleoyl-CoA (0.2-0.3 nmol) and decreases as the acyl-CoA amount increases. Labeled lysophosphatidylcholine (almost exclusively esterified at position 2) is also observed, particularly when the [1-14C]oleoyl-CoA concentration is higher than 0.2-0.3 nmol/50 microliters. The labeled acyl group is mainly inserted in position 2] of the glycerophosphorylcholine. With 0.15 nmol labeled oleoyl-CoA, phosphatidylcholine acylation increases as a function of the protein amount and reaches 25% of the added label at 40 microgram proteins. It is evaluated that, in the presence of 10 microgram proteins, 2% of the microsomal phosphatidylcholine molecules are acylated from 0.1 nmol acyl-CoA. The acylation mechanism seems to involve an acyl exchange between acyl-CoA and phosphatidylcholine.

Effect of cerulenin on the synthesis of very-long-chain fatty acids in microsomes from leek seedlings.

Cerulenin inhibits the elongation of stearoyl-CoA and eicosanoyl-CoA by microsomes from leek seedlings. The inhibition depends on the cerulenin concentration and affects the biosynthesis of docosanoic and tetracosanoic acids only slightly more than that of eicosanoic acid. A 30-min preincubation of the microsomes with cerulenin allows a quantitative inhibition of the elongation at 50 microM cerulenin (50% inhibition at 15 microM cerulenin). A kinetic study of the elongating activity in the presence or in the absence of the inhibitor suggests that the inhibition is non-competitive. Analysis of the products of the reaction suggests that 3-ketoacyl-CoA synthase is the target of cerulenin. A study of the partial reactions demonstrates that the inhibition affects almost exclusively the condensation step.

Yeast mutants affected in viability upon starvation have a modified phospholipid composition.

Selection of mutations, based on the suppression of rvs161 delta defects, was performed. Ten mutants were obtained, ranged amongst four complementation groups, named SUR1, SUR2, SUR3 and SUR4. All sur mutations also suppress a mutation in another gene, RVS167, indicating that all six genes are involved in the same biological pathway. The sur mutant cells have abnormal morphologies in stationary phase, i.e. dumbbell-like in sur1, sur2 or sur3 strains and multi-budded in sur4 strains. Several phenotypic characteristics of the physiological suppressors as well as the rvs161 delta strain itself led us to analyse the phospholipid composition of the mutants. The assays show an overall decrease of the phospholipid amounts and modifications in the relative contents of some phospholipid classes in sur mutant cells.

Molecular basis for low temperature compartment formation by transitional endoplasmic reticulum of rat liver.

The molecular basis for temperature compartment formation was investigated using a cell-free system from rat liver. The donor was from liver slices prelabeled with [3H]acetate. Unlabeled Golgi apparatus membranes were immobilized on nitrocellulose as the acceptor. When transfer was determined as a function of temperature, a transition in transfer activity was observed at low temperatures (less than or equal to 20 degrees C) similar to that seen in vivo. The decrease in transfer efficiency correlated with a decrease in phosphatidylethanolamine and phosphatidylserine content of the transition vesicles formed. By adding lipid mixtures enriched in these lipids to the vesicles, their ability to fuse with the cis Golgi apparatus was reconstituted. These findings provide evidence for a role for lipids in low temperature compartment formation.

Phospholipid acylation by mouse sciatic nerve microsomes.

The partition of 0.3 nmol of [1-14C]oleoyl-CoA in the microsomes (10 micrograms proteins) from mouse sciatic nerves is unaffected by the presence of lysophospholipids and is about 45% of the total oleoyl-CoA (77% of the acylglycerophosphocholine partition in the membrane). The concentration of both oleoyl-CoA and acylglycerophosphocholine is over 1 mM in the membrane. There is a selective acyl transfer from acyl-CoA to lysolipid acceptors (oleoyl greater than myristoyl, palmitoyl, stearoyl much greater than eicosanoyl greater than docosanoyl, tetracosanoyl). The exogenous acyl acceptors are acylglycerophosphocholine and acylglycerophosphoinositol and to a lesser extent acylglycerophosphoethanolamine, but not acylglycerophosphoserine. A PC formation from acylGPC in the absence of exogenous acyl donors or from oleoyl-CoA in the absence of exogenous acyl acceptor was also observed.

Interaction of amphiphilic substrates (acyl-CoAs) and their metabolites (free fatty acids) with microsomes from mouse sciatic nerves.

We have measured the partition of stearoyl-CoA and oleoyl-CoA between an aqueous phase and the microsomes from mouse sciatic nerves. A method of microultracentrifugation was used which allowed us to study separately the aqueous phase and the biological membranes. We observed that the partition is dependent upon the amount of acyl-CoAs and membrane proteins but seems to be independent of time. A theoretical analysis of these data allowed interpretation of the binding and release in terms of acyl-CoA surface density in the vesicles. We have also analyzed the fate of the membrane-bound acyl-CoAs. We show that, whereas the apparent partition does not seem to vary, the hydrolysis of the membrane-bound acyl-CoAs followed by the release of free fatty acids from the membrane leads to a modification of the partition of acyl-CoAs between the membrane and the aqueous phase. We propose that there is a constant partition of the aliphatic chains (acyl-CoAs + free fatty acids).

Phospholipid and fatty acid composition of erythrocytes in type I and type II diabetes.

Different data have been reported concerning modifications of the erythrocyte lipid composition in the different types of diabetes. The heterogeneity of diabetes could be a cause for such differences. Ten type I and ten type II diabetics were carefully selected. The patients were poorly controlled (the mean glycosylated hemoglobin was 12.8% +/- 0.7%); their mean age was 54 +/- 5 years, with a mean duration of diabetes of 18 +/- 4 years. One half of them had severe diabetic complications (nephropathy, retinopathy, and/or polyneuropathy). The diabetics were compared with ten controls. The phospholipid composition was determined by HPTLC analysis, and the fatty acid moieties of the total phospholipids were measured by gas liquid chromatography associated with mass spectrometry. Under well-defined experimental conditions, these results demonstrated a slight, but significant (P less than .05), increase in the phosphatidylethanolamine (PE)/phosphatidylserine (PS) ratio using a Ninhydrin quantitation method; there was also an increase in two minor lipids content (phosphatidylinositol, phosphatidic acid) and the appearance of a lysolipid (lysoPE) in the patients. Whatever the type of diabetes, the red blood cells of diabetics showed no significant differences in their fatty acid contents.

Increased acylation of lysophosphatidylcholine in microsomes of trembler mouse peripheral nerves.

We report here the presence of a Stearoyl-CoA: [1-palmitoyl]-glycerophosphorylcholine (LPC) transacylase activity in the microsomal fraction of normal and Trembler mouse sciatic nerves. Under the experimental conditions studied as a function of incubation time, protein concentration, acyl-CoA and LPC concentrations, the transacylase specific activity was 2-3 times higher in the microsomes of the mutant's nerves than in those of the control. The addition of 5 mM ATP-Mg to the incubation medium, in the absence of bovine serum albumin, leads to a 90% decrease of the stearoyl-CoA thioesterase activity, but increases the transacylation by only 10-20%. This may be due to the low value (10 microM) of the apparent K(m) for C(18)-CoA observed for the mutant's transacylase. In microsomes from control nerves, transacylation requires exogenous LPC, whereas in Trembler mouse sciatic nerve microsomes, the transacylase can use endogenous LPC.

Correlation between the morphology and the lipid and protein compositions in the peripheral nervous system of individual 8-day-old normal and trembler mice.

The hereditary, hypertrophic interstitial neuropathy which afflicts the trembler mouse manifests itself about two weeks after birth. Consequently, the identification of these mutant mice was not possible before this age, except when double mutants were available. We show that the trembler mice can be easily distinguished from their normal littermates before the clinical symptoms appear by using an HPTLC/densitometry technique that allows the simple and rapid analysis of the polar lipids extracted from one sciatic nerve. The results presented in this paper demonstrate important differences between the polar lipid compositions of sciatic nerves from 8-day-old normal and trembler littermates, whose phenotypes were confirmed by the morphological analysis of the contralateral sciatic nerves. The small amount of material that is needed for this identification makes it possible to use the remaining nerve material for other studies. Furthermore, important differences between the sciatic nerve protein compositions of normal and trembler mice, identified according to their polar lipid composition, were also observed and these differences can, therefore, also be employed for the identification of the mutants before the manifestation of the clinical symptoms of the trembler neuropathy.

A quantitative developmental study of the peripheral nerve lipid composition during myelinogenesis in normal and trembler mice.

The quantitative evolution of 10 polar lipids was examined in the sciatic nerves of normal and trembler mice between the ages of 3 days and 60 days. In normal nerves, the polar lipids accumulated slowly until the age of 9 days. A period of rapid accumulation then took place until 18 days of age, after which the phospholipids plateaued, while the glycolipid content continued to increase at a slower rate. The results obtained for the sciatic nerves of trembler mice show that the accumulation of all the polar lipids studied, except phosphatidylcholine and hydroxysulfatides, is abnormal from the earliest stages of postnatal development, and strongly support the view that the primary disorder in the trembler peripheral nervous system is one of dysmyelination. With the exception of cardiolipin, all the lipids in the trembler nerves stopped accumulating at the age of 18 days. The cerebrosides were the lipids the most affected severely at all ages.

A quantitative developmental study of neutral lipids during myelinogenesis in the peripheral nervous system of normal and trembler mice.

The quantitative accumulation of neutral lipids during the period of myelination in the peripheral nervous system was studied in normal and trembler mouse sciatic nerves, between the ages of 5 and 27 days. Neutral lipids were resolved by high-performance thin-layer chromatography, using the solvent mixture hexane/diethyl ether/acetic acid (90:15:2, v/v/v). The lipids were quantitated, after copper acetate/phosphoric acid charring, by densitometric scanning, using an external standard technique. Cholesterol and triacylglycerols accumulated in normal nerves throughout the period studied, while cholesteryl esters were not observed at any age. In trembler nerves, the accumulation of cholesterol took place at a much lower rate than in normal nerves and this lipid was deficient from the earliest stages of development. Triacylglycerols were not significantly deficient in trembler nerves during the first 2-3 weeks, but, after the age of 18 days, their quantity diminished significantly. Cholesteryl esters were first detected in the mutant nerves at the age of 18 days. These results, in agreement with those of a previous developmental study of the polar lipids, are strongly in favour of the view that the trembler mutation directly induces a process of dysmyelination and that demyelination is a secondary event.

Nature of the reaction product of [1-14C]stearoyl-CoA elongation by etiolated leek seeding microsomes.

The elongation of [1-14C]stearoyl-CoA by microsomes from etiolated leek seedlings, in the presence of malonyl-CoA and NADPH, has been studied at different substrate and enzyme concentrations. The HPTLC analysis of the whole reaction mixture, followed by the analysis of the label in the fatty acid methyl esters of long-chain acyl-CoAs, phosphatidylcholine (PC), and neutral lipids, showed that the acyl-CoA fraction contained most of the labeled very-long-chain fatty acids. The very-long-chain fatty acids were rapidly formed and released from the elongase(s) as acyl-CoAs. The label of long-chain acyl-CoAs increased for 20 min and then decreased, whereas it increased in PC. Labeled very-long-chain fatty acids appeared in the neutral lipid + free fatty acid fraction after a 20-min lag.

Assay of long-chain acyl-CoAs in a complex reaction mixture.

A method has been developed which allows the quantitative analysis of labeled or unlabeled acyl-CoAs in complex reaction mixtures. The method is based on (a) a quantitative solubilization of acyl-CoAs and lipids, directly in the reaction vessel, by 0.05 M Tris-HCl, pH 7.5/CHCl3/CH3OH (1/3/3, v/v/v); (b) monodimensional TLC of aliquots of the whole reaction mixture, resolving malonyl-CoA, acetyl-CoA, long-chain acyl-CoAs, polar lipids and neutral lipids plus free fatty acids; and (c) quantitation by TLC densitometry and/or TLC radiochromatography. All fractions--and particularly long chain-acyl-CoAs--can then be analyzed for distribution and label of fatty acyl moieties.

Anticooperative binding of L-tryptophan to tryptophanyl-tRNA synthetase from beef pancreas. Study at equilibrium by dialysis and changes in spectroscopic properties.

Equilibrium dialysis and gel filtration studies show that tryptophanyl-tRNA synthetase from beef pancreas binds two molecules of L-tryptophan per dimer in an anticooperative way. The binding of tryptophan ellicits a series of spectroscopic changes in the protein as seen by absorbance, fluorescence and circular dichroism. The molar absorption change of the protein-tryptophan system upon formation of the complex is delta epsilon292 = 10 400 +/- 1000 M(-1) cm(-1) per dimer. Taking an initial symmetrical dimeric protein the two dissociation constants for tryptophan at pH 8, 25 degrees C are respectively K1 = 2.0 +/- 0.5 muM and K2 = 10 +/- 4 muM. They are respectively K1 = 1 +/- 0.25 muM and K2 = 20 +/- 8 muM if one considers a sequenced binding of the two tryptophan molecules. The dichroic band at 290 nm of the free protein disappears when tryptophan is bound. All observed changes are characteristic of tryptophan perturbation and none of tyrosine perturbation. They all exceed the effect that can be expected from the change in environment of the bound tryptophan molecules and modifications of the tertiary structure of the protein have to be taken into account to explain the observed spectroscopic data.