Biosynthesis of very long chain fatty acids in higher plants.
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Acyl-Carrier Protein S-Malonyltransferase↗
Biomedical subjects
C Cassagne
Publications and source records attributed to C Cassagne.
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.
Acetyltransferases↗
Occurrence and characterization of a dehydratase enzyme in the leek icosanoyl-CoA synthase complex.
The presence of a beta-hydroxyacyl-CoA dehydratase involved in the icosanoyl-CoA synthase (EC 2.3.1.119) complex of leek epidermis has been demonstrated using antibodies raised against the purified beta-hydroxyacyl-CoA dehydratase from rat liver. In a first step the leek icosanoyl-CoA synthase activity was measured in the presence of different amounts of this antibody, the results obtained showed a 75% inhibition of the activity using a 8:1 IgG/microsomal protein ratio, whereas only a weak diminution of the activity occurred using pre-immune IgG. The analysis of the reaction products after incubation in the presence of increasing IgG amounts showed a decrease of the fatty acids (the final product) and an accumulation of beta-hydroxy fatty acids using immune IgG, whereas no change occurred in the presence of pre-immune IgG. Moreover, the beta-hydroxyacyl-CoA dehydratase activity was strongly inhibited, whereas in the same conditions, the beta-ketoacyl-CoA reductase and the (trans-2-3) enoyl-CoA reductase activities were not affected. The protein fractions that eluted from the DEAE and Ultrogel columns containing the leek icosanoyl-CoA synthase activity were able to specifically bind the anti beta-hydroxyacyl-CoA dehydratase from rat liver. The cross-reactivity was demonstrated. In immunoblotting experiments using the same antiserum after SDS-PAGE of the purified leek icosanoyl-CoA synthase, only one of the four protein bands constituting the leek icosanoyl-CoA synthase was detected. This protein, having an apparent molecular mass of 65 kDa, could be the dehydratase component of the elongation complex.
Allium↗
Ceramide excluded from cell-free vesicular lipid transfer from endoplasmic reticulum to Golgi apparatus. Evidence for lipid sorting.
The distribution and cell-free transfer of ceramide and other lipids were compared using highly purified fractions of endoplasmic reticulum, transitional endoplasmic reticulum, transition vesicles and Golgi apparatus from rat liver. Ceramides were present in both endoplasmic reticulum and Golgi apparatus where they represented between 0.3 and 1% of the total lipids. Ceramides, however, were much reduced or absent (< 0.05%) from transition vesicles. Transition vesicles were induced to form from transitional endoplasmic reticulum by incubation with ATP and a cytosol fraction. When transfer of [14C]choline-labeled phosphatidylcholine from transitional endoplasmic reticulum to Golgi apparatus was followed, transition vesicles were more efficient in transfer than the transitional endoplasmic reticulum from which they were derived. This transfer was temperature- and ATP-dependent and inhibited by N-ethylmaleimide. When transfer of [3H]ceramide was followed, there was little or no transfer via transition vesicles and that transfer which occurred was temperature-, ATP- and N-ethylmaleimide independent. Transfer of ceramide in the cell-free system did occur from endoplasmic reticulum to Golgi apparatus but via a non-vesicular mechanism that was temperature-dependent but not dependent on ATP or cytosol, alone, or in combination, nor was it inhibited by N-ethylmaleimide. A component of phosphatidylcholine transfer exhibited similar characteristics. The results provide evidence for two distinct mechanisms for cell-free transfer of lipids from endoplasmic reticulum to Golgi apparatus. The first is via 50 to 70 nm transition vesicles which is temperature- and ATP-dependent, inhibited by N-ethylmaleimide and from which ceramides are excluded. The second is non-vesicular, temperature-dependent, and neither ATP- nor cytosol-dependent. It accounts for the bulk of the ceramide transfer. As a result during cell-free lipid transfer from endoplasmic reticulum to Golgi apparatus, lipid sorting occurs such that ceramides are largely absent from the transition vesicles and, apparently are delivered to the Golgi apparatus by another mechanism.
Animals↗
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.
Cell Division↗
Demonstration that anti-phospholipid auto-antibodies react with both anionic and zwitterionic phospholipids.
The specificity of anti-phospholipid auto-antibodies present in the serum of systemic lupus erythematosus patients towards the phospholipids has been studied by two different methods. The antibodies have been characterized either after affinity purification or by inhibition experiments. Our results clearly demonstrate that anti-phospholipid auto-antibodies recognize all phospholipids, whatever their polar head.
Anions↗
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.
Adenosine Triphosphate↗
Evaluation of the amount of acyl-CoA elongases in leek (Allium porrum L) leaves.
Polyclonal antibodies have been raised against the acyl-CoA elongase purified from leek epidermal cells. The antibodies recognize the fractions containing the elongating activity after DEAE or Ultrogel chromatography and their response with the other fractions is very low. The immune complex is immunoprecipitable with Protein A-Sepharose. 1% of the solubilized proteins from leek epidermis microsomes are immunoprecipitated. The immunoprecipitate contains an elongating activity which is 86 +/- 20-times that of the unbound fraction.
Acyl-Carrier Protein S-Malonyltransferase↗
Solubilization and partial purification of constituents of acyl-CoA elongase from Lunaria annua.
All the constituent enzymes of acyl-CoA elongase, i.e., beta-ketoacyl-CoA synthase, beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase, have been solubilized from a 15,000 x g particulate fraction from developing seeds of honesty (Lunaria annua) using Triton X-100. All these activities were retained upon subsequent precipitation of the solubilized protein with polyethylene glycol and resuspension of the precipitate followed by ion exchange chromatography of the resulting protein on DEAE-cellulose. A 4.2-fold enrichment of the acyl-CoA elongase was thus obtained. Further chromatography of the DEAE fraction containing all the constituents of acyl-CoA elongase on Ultrogel yielded a major protein fraction exhibiting the activities of beta-ketoacyl-CoA synthase and beta-ketoacyl-CoA reductase only. Almost 30-fold purification of the beta-ketoacyl-CoA synthase was thus achieved. The beta-ketoacyl-CoA synthase was inhibited only at high concentrations of cerulenin, but at very low concentrations of iodoacetamide. Inhibition could be reduced by preincubation with thioesters, indicating that an enzyme thioester intermediate is involved in the condensation reaction of the acyl-CoA elongation.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Fatty acids rendered immunogenic.
A method allowing the covalent binding of fatty acids to a carrier was developed. The absence of absorbed fatty acids was controlled by HPTLC. Rabbits were immunized with these immunogens. The antibodies obtained were studied by ELISA and were shown to be directed against the fatty acyl chain, whatever its length.
Animals↗
Lack of in vitro ceramide formation in the PNS of trembler mice.
The amidification of sphingosine by acyl donors has been investigated in a microsomal fraction prepared from sciatic nerves of normal and Trembler mice. In the control, a ceramide synthesis is observed in the presence of acyl-CoAs and not with free fatty acids. The synthesis increases as a function of the protein amount and the time and is dependent on acyl-CoA concentration. The level of synthesis is highly similar to that observed in vivo after palmitate injection into the sciatic nerves of normal mice. In the mutant, there is a major abnormality because a weak synthesis (20% of the control) is observed only with high acyl-CoA concentration (greater than 200 microM), whereas in the range of the physiological acyl-CoA concentrations (less than 20 microM), there is no ceramide formation from stearoyl-CoA or lignoceroyl-CoA.
Acyl Coenzyme A↗
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.
Acylation↗
Monensin blocks the transfer of very long chain fatty acid containing lipids to the plasma membrane of leek seedlings. Evidence for lipid sorting based on fatty acyl chain length.
Delivery of newly synthesized fatty acids and lipids to the plasma membrane in leek seedlings via the endoplasmic reticulum (ER)-Golgi apparatus pathway is primarily by bulk transport (without sorting). However, pulse-chase experiments revealed kinetics of transport of lipids with VLCFA (very long chain fatty acids having more than 18 carbon atoms) in favor of a preferential transfer of these molecules to the plasma membrane. Use of monensin showed the accumulation of lipids in the Golgi apparatus and a related decrease of the amount of lipids transported to the plasma membrane. Lipid and fatty acid analyses revealed that transport of VLCFA-containing phospholipids was most strongly inhibited by the monensin block. These results taken together with an inability of the plasma membrane to synthesize VLCFA support a role for the Golgi apparatus in VLCFA delivery to the plasma membrane and leads to the hypothesis of a sorting function as well, based on fatty acyl chain length.
Allium↗
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).
Acyl Coenzyme A↗
Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver.
Trafficking and sorting of lipids during transport from the endoplasmic reticulum to the Golgi apparatus was studied using a cell-free system from rat liver. Transitional elements of the endoplasmic reticulum were prepared from liver slices prelabeled with [14C]- or [3H]acetate as the donor fraction. Non-radioactive Golgi apparatus were immobilized on nitrocellulose as the acceptor. When reconstituted, the radiolabeled donor retained a capacity to transfer labeled lipids to the non-radioactive Golgi apparatus acceptor. Transfer exhibited two kinetically different components. One was stimulated by ATP, facilitated by cytosol and inhibited by guanosine 5'-O-(thiotriphosphate) and N-ethylmaleimide. In parallel with protein transport, the ATP-dependent lipid transfer occurred with a temperature transition at about 20 degrees C. The other was not stimulated by ATP, did not require cytosol, was acceptor unspecific, was unaffected by inhibitors and, while temperature dependent, did not exhibit a sharp temperature transition. The ATP-independent transfer was non-vesicular. In contrast, the ATP-dependent transfer was vesicular. Transition vesicles isolated by preparative free-flow electrophoresis, when used as the donor fraction, transferred lipids to Golgi apparatus acceptor with a 5-6-fold greater efficiency than that exhibited by the unfractionated transitional endoplasmic reticulum. Formation of transition vesicles was ATP-dependent. Transferred lipids were chiefly phosphatidylcholine and cholesterol. Membrane triglycerides, major constituents of the transitional endoplasmic reticulum membranes, were both depleted in the transition vesicle-enriched fractions and not transferred to Golgi apparatus suggestive of lipid sorting prior to or during transition vesicle formation. The characteristics of the ATP plus cytosol-dependent transfer were similar to those for protein transfer mediated by transition vesicles. Thus, the 50-70-nm vesicles derived from transitional endoplasmic reticulum appear to function in the trafficking of both newly synthesized proteins and lipids from the endoplasmic reticulum to the Golgi apparatus.
Adenosine Triphosphate↗
Autoantibodies against all the phospholipids: a comparative systematic study with systemic lupus erythematosus and healthy sera.
The sera of systemic lupus erythematosus patients were tested by ELISA for the presence of autoantibodies against all the phospholipids: cardiolipin, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylserine. The quantity of phospholipid coated (in comparison with that initially deposited) on the microtitration plates was precisely evaluated in order to determine if the results (absorbance values) obtained for each phospholipid could be compared directly. The systemic lupus erythematosus sera tested gave positive results for all the phospholipids. The highest level of autoantibodies was observed with phosphatidic acid followed by phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. The sera seemed to contain antibodies directed either against all the 8 phospholipids tested or more specifically against one or two phospholipids. The results were compared with those obtained with 17 healthy sera. Much lower values were obtained for the sera of healthy subjects, the majority of which showed a weak binding, similar for all the phospholipids. These results suggest that the anti-phospholipid autoantibodies present in systemic lupus erythematosus sera are significantly higher than those of healthy subjects. It is concluded that in the investigation of anti-phospholipid antibodies, tests should be carried out for all the phospholipids.
Antibody Specificity↗
Normal composition of the major lipids and the distribution of their fatty acids in central nervous system myelin further discriminate mld from shiverer mice.
The primary defect in myelin deficiency (mld), an autosomal recessive mutation in mice with severe hypomyelination of the central nervous system (CNS), is a reduction in the synthesis of myelin basic protein (MBP) due to reduced amounts of MBP-specific mRNA. The present study was performed to determine whether alterations of myelin lipid composition might be associated with this defect. Although a 20-fold reduction of myelin was found in mld brain, the lipid/protein and the phospholipid/sulfatide ratios of purified myelin were unchanged. The analysis of polar lipids demonstrated no striking difference. However, phosphatidylethanolamine (including plasmalogens of the ethanolamine type) was decreased by 7.7% in mld. Fatty acids of total and polar lipids (phospholipids + glycolipids) exhibited a slight decrease in 20:1(n-11) and 20:1(n-9). In conclusion, these results further differentiate mld from its allele shiverer, which shares with mld a dramatic reduction of MBP and absence of major dense line but, in contrast, presents other important biochemical differences in CNS myelin.
Animals↗
Specific immunocytochemical visualization of phosphatidylserine.
In order to determine whether anti-phosphatidylserine antibodies are able to detect phosphatidylserine in situ, an immunocytochemical approach has been developed using human platelets. A strong positive reaction was obtained when anti-phosphatidylserine serum was applied to platelets whereas no reaction was observed with preimmune serum, or with immune serum which had been preincubated with 10(-5) M phosphatidylserine, suggesting that phosphatidylserine was indeed specifically detected by the antibodies.
Blood Platelets↗