Acylation of dog heart lysophosphatidylserine by transacylase activity

Dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of phosphatidylcholine (PC) to lysophosphatidylserine (lysoPS) in the presence of coenzyme A (CoA) at pH optima of 4.5-5.0 and 7.5. Acyl transfer activity at acidic pH is about three times higher than at neutral pH. Transacylation of lysoPS by acyl transfer from PC with dog heart microsomes at neutral pH favors arachidonate over linoleate by a factor of 2.1, whereas free linoleic acid is favored by a factor of 3.7 over arachidonic acid for lysoPS acylation in the presence of acyl-CoA-generating cofactors. Considering the location and acyl composition of myocardial PS, it appears that both acyl transfer from PC and utilization of unesterified fatty acids may be involved in the acylation of lysoPS at its sn-2 position.     

Selectivity of acyl transfer between phospholipids: arachidonoyl transacylase in dog heart membranes

Dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of exogenous phosphatidylcholine to 1-acyl lysophosphatidylethanolamine. Approximately equal amounts of free fatty acids are produced as well. The reaction exhibits a pH optimum of 7.5-8.5 and does not require Ca2+ or other divalent cations. The reaction proceeds in the absence of exogenous coenzyme A but acyl transfer is enhanced by its addition. The transacylase exhibits a strong preference for arachidonate over linoleate and thus may be involved in the maintenance of the high amounts of arachidonate found in microsomal ethanolamine phospholipids.     

Curvature-dependent recognition of ethanolamine phospholipids by duramycin and cinnamycin

Duramycin is a 19-amino-acid tetracyclic lantibiotic closely related to cinnamycin (Ro09-0198), which is known to bind phosphatidylethanolamine (PE). The lipid specificity of duramycin was not established. The present study indicates that both duramycin and cinnamycin exclusively bind to ethanolamine phospholipids (PE and ethanolamine plasmalogen). Model membrane study indicates that the binding of duramycin and cinnamycin to PE-containing liposomes is dependent on membrane curvature, i.e., the lantibiotics bind small vesicles more efficiently than large liposomes. The binding of the lantibiotics to multilamellar liposomes induces tubulation of membranes, as revealed by electron microscopy and small-angle x-ray scattering. These results suggest that both duramycin and cinnamycin promote their binding to the PE-containing membrane by deforming membrane curvature.     

Covalent binding of isoketals to ethanolamine phospholipids

Free radicals have been strongly implicated in the pathogenesis of many human diseases. We previously identified the formation of highly reactive gamma-ketoaldehydes, isoketals, in vivo as products of free radical-induced peroxidation of arachidonic acid. Isoketals react with lysine residues on proteins at a rate that far exceeds that of 4-hydroxynonenal and demonstrate a unique proclivity to crosslink proteins. Hydroxynonenal has been shown to react with aminophospholipids, particularly phosphatidylethanolamine. We explored whether isoketals also react with phosphatidylethanolamine. Using liquid chromatography/electrospray mass spectrometry, we found that isoketals form pyrrole and Schiff base adducts with phosphatidylethanolamine. In addition, the ability of isoketals to covalently modify phosphatidylethanolamine is greater than that of 4-hydroxynonenal. These studies identify in vitro novel isoketal adducts. This provides the basis to explore the formation of isoketal-aminophospholipid adducts in vivo and the biological consequences of the formation of these adducts.     

N-Acylation of ethanolamine phospholipids by acyl transfer does not involve hydrolysis

N-Acylethanolamine phospholipids occur in infarcted but not in normal canine myocardium. Their synthesis is catalyzed by a membrane-bound, Ca2+-requiring N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of various phospholipids including phosphatidylethanolamine to the amino group of ethanolamine phospholipids. When dog heart mitochondria are incubated in media containing Ca2+ and H2(18)O, the resulting N-acylethanolamine phospholipids do not accumulate 18O in either the amide or 1-O-acyl groups. The results indicate that acyl transfer occurs without hydrolysis, most likely through an acyl-enzyme complex which may be covalently linked.     

Replacement of ethanolamine phosphate by 3-aminopropylphosphonate in the phospholipids of Tetrahymena

When Tetrahymena thermophila is grown on a medium containing up to 5 mm 3-aminopropylphosphonate, up to 90% of the ethanolamine phosphate in phosphatidylethanolamine is replaced by the 3-aminopropylphosphonate. No accompanying alteration of the phospholipid composition of Tetrahymena is observed. This contrasts with the results obtained when 2-aminoethylphosphonate, the naturally occurring compound, is added to the growth medium (Biochim. Biophys. Acta528, 394–398, 1978); the 2-aminoethylphosphonate causes a substantial increase in the 2-aminoethylphosphonolipid and a reciprocal decrease in phosphatidylethanolamine. Thus, there is apparently a one-way control system in Tetrahymena whereby 2-aminoethylphosphonate and its phosphonolipid may influence the level of phosphatidylethanolamine in the cell, but ethanolamine phosphate, as represented by its isosteric analog, does not influence the phospholipid levels. There is no effect of the 3-aminopropylphosphonate on de novo 2-aminoethylphosphonate biosynthesis indicating a strict specificity for 2-aminoethylphosphonate as its own feedback inhibitor.     

Coenzyme A-dependent and -independent acyl transfer between dog heart microsomal phospholipids

We have recently shown that dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of exogenous phosphatidylcholine to lysophosphatidylethanolamine with strong preference for arachidonate over linoleate (Biochem. Biophys. Res. Commun. 129, 381-388 (1985)). We now report that the addition of 0.5 mM CoA enhances the acyl transfer activity 3-4-fold but reduces the selectivity for arachidonate. Acyl transfer in the absence of CoA exhibits a pH optimum of 7.5-8.5, whereas two pH optima (7.5 and 4.5) are observed in the presence of CoA with transfer activity at pH 4.5 exceeding that of pH 7.5 by 4-5-fold. The plasmalogen (alkenyl) analog of lysophosphatidylethanolamine is an equally effective acyl acceptor in the absence of CoA but less effective in its presence. The microsomal acyl-CoA/lysophosphatidylethanolamine acyltransferase does not favor arachidonate over linoleate. Therefore, transacylation from phosphatidylcholine may account for the high arachidonate content of dog heart microsomal phosphatidylethanolamine and its plasmalogen analog. In fact, acyl transfer from endogenous lipids to 1-[1'-14C]palmitoyl-2-lyso-sn-glycerophosphoethanolamine results in the generation of mostly (over 80%) tetraunsaturated phosphatidylethanolamine. This proportion is reduced by the addition of CoA and, even more, by CoA plus acyl-CoA-generating cofactors. We conclude that in dog heart microsomes, lysophosphatidylethanolamine can be acylated by different mechanisms, of which the CoA-independent transacylase exhibits the greatest acyl selectivity.     

Quantification of choline and ethanolamine phospholipids in rabbit myocardium

To facilitate evaluation of the influence of myocardial phospholipid metabolites on the development of electrophysiologic abnormalities induced by ischemia, a method for the quantification of choline and ethanolamine phospholipids suitable for accurate and reproducible analysis of small amounts of myocardium was developed. The procedure combines chloroform and methanol extraction of phospholipids after tissue homogenization with subsequent separation by sequential thin-layer and high-performance liquid chromatography. Phosphorus in purified lipid classes was determined with the correction for recovery based on 14C-labeled internal standards.     

Biosynthesis of N-acylethanolamine phospholipids by dog brain preparations

Abstract: 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 (∼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 glycero-phosphocholine 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.     

Incorporation of choline and ethanolamine into phospholipids in germinating soya bean.

1. Incorporation of [Me-14C]choline and [2-14C]ethanolamine into lipids was studied in germinating soya bean (Glycine max L.) seeds. The precursors are only incorporated into phosphatidylcholine and into phosphatidylethanolamine respectively. 2. Base-labelling via a phospholipase-D type of reaction was eliminated as a significant factor. 3. Cyclo heximide inhibited labelling of phosphatidylcholine from [Me-14C]choline but did not affect labelling of the aqueous choline pool. It had no effect on [2-14C]ethanolamine uptake or incorporation into phosphatidylethanolamine. 4. Hemicholinium-15 at 10mM concentrations decreased uptake and lipid labelling from the both bases. 5. There was no evidence for base competition. 6. The endogenous pool of choline was much larger than that of ethanolamine, which resulted in higher specific radioactivities for phosphatidyl-ethanolamine than for phosphatidylcholine. 7. The results can be interpreted as indicating that the kinase and phosphoryltransferase enzymes of the CDP-base pathways are separate for each phospholipid.     

Incorporation of serine and ethanolamine into the phospholipids in rabbit retina.

The incorporation of serine and ethanolamine into phospholipids in rabbit retinal subcellular fractions and in excised retinas was studied in vitro, and some enzymic properties of the incorporation of phospholipid bases by base exchange were examined in the microsomal fraction. The retina was found to have a higher rate of base exchange for the incorporation of phospholipid bases than other tissues. The retinal microsomal fraction possessed the highest specific activity of base exchange, while the rod outer segment had very little activity. These results suggest that the phospholipids in the rod outer segment may be transferred from the inner segment of the photorecepter cell. The apparent Km values for serine and ethanolamine in the microsomal fraction decreased with decreasing Ca2+ concentration. Although no further increase of incorporation of serine and ethanolamine occurred after 40 min in the microsomal fraction, continuous incorporation of both bases into phospholipids was seen for 3 hr in excised retina. Illumination did not significantly affect the incorporation of serine and ethanolamine in excised retina or in the rod outer segment fraction. Base exchange reaction thus may not play a direct role in the visual process.     

Isolation of very low density lipoprotein phospholipids enriched in ethanolamine phospholipids from rats injected with Triton WR 1339

Phospholipids carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases and lecithin-cholesterol acyltransferase. We have previously demonstrated [J.J. Agren, A. Ravandi, A. Kuksis, G. Steiner, Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis, Eur. J. Biochem. 269 (2002) 6223-6232] that the infusion of Triton WR 1339 (TWR), which inhibits these lipases, leads in 2 h to five-fold increase in VLDL triacylglycerol concentration along with major differences in the composition of their molecular species. The present study demonstrates that the accumulation of triacylglycerols is accompanied by major changes in the content of the VLDL phospholipids, of which the most significant is the enrichment of phosphatidylethanolamine (PtdEtn). This finding coincides with the enrichment in PtdEtn demonstrated in the VLDL of a hepatocytic Golgi fraction but it had not been demonstrated that the Golgi VLDL, along with its unusual phospholipid composition, can be directly transferred to plasma. Aside from providing an easy access to nascent plasma VLDL, the TWR infusion demonstrates that lipoprotein and hepatic lipases are also responsible for the degradation of plasma VLDL PtdEtn, as independently demonstrated for plasma phosphatidylcholine. Our results indicate also, with the exception of lysophosphatidylcholine, that preferential basal hydrolysis no dot lead to major differences in molecular species composition between circulating and newly secreted VLDL phospholipids. The comparison of the molecular species composition of VLDL and liver phospholipids suggests a selective secretion of PtdEtn and sphingomyelin molecular species during VLDL secretion.     

sn-Glycerol-3-phosphate transacylase activity in Euglena gracilis organelles

sn-Glycerol-3-phosphate transacylase activity was demonstrated in Euglena mitochondria, chloroplasts, and microsomes. There was no activity in the 100,000g 1-h supernatant. Exposure of each of the isolated organelles to 1 × 10^?4% Triton X-100 resulted in release of substantial quantities of transacylase activity into the 100,000g supernatant. Products formed by catalysis by the membrane-bound transacylases were heterogenous, while those resulting from catalysis by the extracted enzymes were practically all lysophosphatidate.     

Modulation of solubilized brain fucosyltransferase activity by phospholipids

Phospholipids interact on Triton X-100 solubilized GDP-fucose: asialofetuin fucosyltransferase (EC 2.4.1.68) isolated from sheep brain. This enzymatic activity is modulated by charged phospholipids. In particular, phosphatidic acid and analogues markedly inhibit the transfer of fucose from GDP-[14C]fucose. Kinetic studies show that phosphatidic acid interacts as a mixed inhibitor: the velocity and affinity of fucosyltransferase for the GDP-fucose and asialofetuin substrates are strongly decreased. However, this inhibitory effect is not related to stereospecificity, and the different parameters involved in the enzymatic reaction of glycosylation are not modified. The nature of fatty acids and chemical bond (ester or ether) occurring in the carbohydrate chain does not modify the behaviour of phosphatidic acid with respect to fucosyltransferase activity. Further, the physical state of phosphatidic acid (gel phase or liquid crystalline phase) has no influence. However, as the inhibition is closely pH-dependent, these data suggest that phosphatidic acid might directly interact with the active site of the enzyme and induce a conformational change.     

Regulation of liver base-exchange activity by acidic phospholipids

Liver microsomes were enriched in liposomal acidic lipids by Ca²⁺-dependent fusion of liposomes at pH 7.0. The extent of fusion was monitored by the transfer of radioactive cholesteryl oleate. The enrichment of membranes in phosphatidylserine inhibited ethanolamine base-exchange, whereas the fusion with phosphatidylinositol inhibited both ethanolamine and serine base-exchange reactions. In contrast, these two phospholipids had scarce effects on choline base-exchange. Phosphatidic acid did not suppress any of the three base-exchange activities. Possible functional implications are discussed.Abbreviations DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - SHB suerose-HEPES buffer (0.25M sucrose, 3mM HEPES, pH 7.4)     

Quantification of surfactant phospholipids in the dog lung

We quantified total phospholipid (PL), total and disaturated phosphatidylcholine (PC and DSPC), phosphatidylglycerol (PG), and total protein in alveolar washings and lung tissue in 22 dog lungs. Quantitative recovery of alveolar material and assessment of its possible contamination by blood lipids were important determinants of methodology. To remove blood, the vessels of half the lungs were perfused with a fluorocarbon emulsion before lavage. The volume of blood removed by perfusion and the quantity and fatty acid patterns of its whole blood and plasma PL and PC were determined. Washings of unperfused lungs contained means of 21% more PL and 24% more PC than those of perfused lungs. Although this excess could be accounted for by the PL and PC in pulmonary blood, the hemoglobin and total protein content of washings and their PC fatty acid patterns indicated that blood lipids were not a major source of the excess lipid in washings of unperfused lungs. Using more recent morphometric estimates rather than the indirect ones previously used by others, the quantity of alveolar DSPC (1 mg/g lung) is calculated to be 1.8 times the amount necessary to form a packed monolayer on the internal surface of the lung at functional residual capacity.