Biosynthesis of N-acylethanolamine phospholipids by dog brain preparations.

Dog brain homogenates and subcellular preparations incubated in the presence of Ca2+ produced a new phospholipid that was isolated and identified by its infrared spectrum and by chemical degradation as a mixture of 1,2-diacyl, alkenylacyl, and alkylacyl sn-glycero-3-phospho(N-acyl)ethanolamines, 50, 45, and 5%, respectively. The N-acyl groups consisted almost exclusively of 16:0, 18:0, and 18:1 fatty acids. Formation of N-acylethanolamine phospholipids from endogenous substrates was linear for about 90 min at approximately 4.5 nmol/h/mg protein and exhibited a pH optimum of 10. Biosynthetic activity was associated with particulate fractions, primarily microsomes, synaptosomes, and mitochondria, but not with myelin. In each case, small amounts (approximately 0.5 nmol/h/mg protein) of long-chain N-acylethanolamines were also produced. Incubation of dog brain microsomes with 1,2-di[1'-14C]palmitoyl glycerophosphocholine yielded N-acylethanolamine phospholipids labeled at both N-acyl (55%) and O-acyl (45%) moieties. It appears that dog brain organelles may contain a phosphatidylethanolamine N-acyl transferase (transacylase) analogous to that recently demonstrated in the myocardial tissue.

The role of cardiolipin as an acyl donor in dog heart N-acylethanolamine phospholipid biosynthesis.

N-Acylethanolamine phospholipids were produced from endogenous substrates with dog heart mitochondrial and microsomal preparations. With mitochondria the N-acyl group contained 13.8% linoleate, with microsomes only 3.6%. Cardiolipin comprised 18.5% of mitochondrial and 3.3% of microsomal lipid P and contained 93.7 and 72.4% linoleic acid, respectively. Incubation of dog heart subcellular fractions with [1-14C]linoleoyl cardiolipin in the presence of Ca2+ resulted in the formation of N-acylethanolamine phospholipids labeled primarily in the N-acyl and 1-O-acyl moieties. The data indicate that cardiolipin is the major source of linoleic acid used in the N-acylation of ethanolamine phospholipids by transacylase activity.

Role of phosphatidylethanolamine in the biosynthesis of pyrophosphoethanolamine residues in the lipopolysaccharide of Escherichia coli.

The biosynthesis of pyrophosphoethanolamine residues linked to the core oligosaccharide region of the lipopolysaccharide of Escherichia coli K2 strain BB 26-36 has been investigated by means of isotope tracer experiments in living cells. Phosphoethanolamine was isolated from the pyrophosphoethanolamine residues after hydrolysis in 1 N HCl at 100 degrees C. The kinetics of labeling of the phosphoethanolamine from [3H]serine or sn-glycero-3-32P during pulse-chase experiments revealed that the biosynthetic precursor of the phosphoethanolamine must be a large, relatively stable pool, and not a small, rapidly metabolized pool such as that of free serine, or seryl-tRNA. Labeling of the pyrophosphoethanolamine residues of the lipopolysaccharide from the two isotopes was closely parallel, and the isotope ratio 3H/32P was closely similar to that in phosphatidylethanolamine at the same time intervals. These experiments offer strong evidence that phosphatidylethanolamine functions in the biosynthesis of pyrophosphoethanolamine residues in lipopolysaccharide in a reaction in which the phosphoethanolamine head-group of the phospholipid is transferred as a unit to a lipopolysaccharide acceptor.

Alteration in enzyme activities of de novo phosphatidylcholine biosynthesis in rat liver by treatment with typical inducers of microsomal drug-metabolizing system.

1. The activities of choline kinase, ethanolamine kinase, cholinephosphate cytidylytransferase(s) and cholinephosphotransferase were compared in the liver subcellular fractions after the treatment of rats for two successive days with phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls. 2. The administration of phenobarbital resulted in a significant decrease in choline kinase activity while not affecting ethanolamine kinase activity. Both 3-methylcholanthrene and polychlorinated biphenyls caused considerable enhancement of choline kinase activity concomitantly with ethanolamine kinase activity. 3. The activity of cytosolic cytidylytransferase was not affected by any of the inducers while the microsomal activity was significantly depressed by the administration of either phenobarbital or polychlorinated biphenyls. 4. The activity of microsomal cholinephosphotransferase decreased significantly after the treatment with both 3-methylcholanthrene and polychlorinated biphenyls and increased slightly after phenobarbital administration. 5. The observed opposite effects of phenobarbital and 3-methylcholanthrene on the enzymes in de novo phosphatidylcholine synthesis indicate that there exist a possible relation between induction of microsomal drug-metabolizing system and modulation of phosphatidylcholine biosynthesis in animal liver.

The action of piracetam on the formation of ethanolamine-plasmalogen by neuronal microsomes of the developing rat brain.

In search of a common biochemical denominator of the action of the nootropic drug 2-oxo-pyrrolidine-1-acetamide (piracetam, Normabrain, Nootrop) the effects of the substance on the neuronal respiratory chain were investigated. The activity of the electron transport system of the respiratory chain was measured by the conversion of the ether to the enolether bond (plasmalogen) of ethanolamine containing glycerophosphatides. Piracetam enhances the formation of ethanolamine-plasmalogen from the corresponding ether lipid by neuronal microsomes and thus resembles the action of cytochrome b5. The addition of antibody against cytochrome b5 was able to inhibit the piracetam-dependent stimulation of the plasmalogen biosynthesis. Thus it appears that the stimulatory effect of piracetam on the formation of ethanolamine-plasmalogen is mediated by an increased synthesis or turnover of cytochrome b5.

Biosynthesis of rat brain phosphatidylethanolamines from intracerebrally injected ethanolamine.

[2-3H]Ethanolamine was injected intracerebrally into male rats and the brains of the animals immediately removed by particular procedures at regular intervals over the first 1200 sec. The incorporation of radioactivity into brain phosphorylethanolamine, cytidine-5'-diphosphate (CDP) ethanolamine and phosphatidylethanolamines was examined and quantitated. The nature of phosphatidylethanolamine molecular subspecies, which became labelled, was also investigated after isotope administration. Phosphorylethanolamine, CDP-ethanolamine and phosphatidylethanolamines were all labelled already 5 sec after the administration of labelled ethanolamine. The specific radioactivities of different phosphatidylethanolamine molecular subspecies varied according to the time elapsed from the injection to the sacrifice of the animals. This last result, together with the data on time course of labelling of ethanolamine phosphoglycerides and their precursors, provides indications that this base may be incorporated into lipids not only by net synthesis pathway, but also by base-exchange reaction.

Replacement of acyl and alk-1-enyl groups in Clostridium butyricum phospholipids by exogenous fatty acids.

The effect of exogenous unsaturated fatty acids on the acyl and alk-1-enyl group composition of the phospholipids of Clostridium butyricum has been examined. Unsaturated fatty acids support the growth of this organism in the absence of biotin. When cells were grown at 37 degrees in media containing oleate or linoleate and a Casamino acid mixture containing traces of biotin, the exogenous fatty acids were found mainly in the alk-1-enyl chains of the plasmalogens with less pronounced incorporation into the acyl chains. However, at 25 degrees in this medium, both the acyl and alk-1-enyl chains contained substantial amounts of the 18:1 supplement plus the C19-cyclopropane chains derived from it. Ak-1-enyl chains in all the major phosphatide classes showed a uniformly high substitution by the oleate supplement in cells grown at 37 degrees. The oleate and C19-cyclopropane content of the acyl chains was more variable among the phosphatide classes. At 37 degrees, trans-9-octadecenoic acid (elaidic acid) also supported growth and was incorporated into both acyl and alk-1-enyl chains at a high level. When cells were grown on oleate at 37 degrees in media containing biotin-free Casamino acids, both the acyl and alk-1-enyl chains had a high level of 18:1 plus C19-cyclopropane chains. In the cells grown at 37 degrees with oleate substantial changes were seen in the phospholipid class composition. There was a large decrease in the ethanolamine plus N-methylethanolamine plasmalogens with a corresponding increase in the glycerol acetals of these plasmalogens. The glycerol phosphoglycerides were also significantly lower with the appearance of an unknown, relatively nonpolar phospholipid fraction.