Regulation of lysophosphatidylcholine-metabolizing enzymes in isolated myocardial cells from rat heart

Enzymatic pathways involved in the metabolism of lysophosphatidylcholine were investigated in rat heart myocardial cells. Acyl CoA-dependent acyltransferase activity was localized in microsomes, and was much greater than lysophospholipase activity in either cytosolic or microsomal fractions. The cytosolic lysophospholipase was more sensitive to inhibition by palmitylcarnitine in comparison to free fatty acids. In contrast, free fatty acids (oleate and palmitate) produced a greater inhibition of the microsomal acyltransferase and lysophospholipase than did palmitylcarnitine. A reduction in the assay pH to 6.5 resulted in an increase in microsomal acyltransferase and cytosolic lysophospholipase activities, but brought about a marked reduction in the microsomal lysophospholipase activity. At pH 6.5, the percentage inhibition of the microsomal acyltransferase by palmitylcarnitine was reduced, whereas the inhibition by palmitic acid was enhanced. The inhibition of the microsomal lysophospholipase by both palmitylcarnitine and palmitic acid was reduced at pH 6.5. With respect to myocardial ischemia, the inhibition of microsomal acyltransferase by free fatty acids and the reduction in microsomal lysophospholipase activity due to acidosis may contribute to the elevation of cellular lysophosphoglycerides which are arrhythmogenic.     

Phospholipase C activity in rat kidney. Effect of deoxycholate on phosphatidylinositol turnover

Rat renal cortical and medullary slices incorporate [14C]arachidonate into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerols. The percent distribution of [14C]arachidonate among the various phospholipids is similar in renal cortex and medulla, although the total amount of radioactively labeled phospholipids is higher in the renal medulla. Subsequent incubation of prelabeled slices in the presence of deoxycholate induces a loss of radioactivity from [14C]phosphatidylinositol, with a concomitant increase in 1,2-[14C]diacylglycerol. Neutral lipids are not affected. The degradation of phosphatidylinositol to [14C]diacylglycerol indicates the presence of phospholipase C activity. Renal medulla seems to be more sensitive to deoxycholate than the renal cortex. Deoxycholate also induces slightly the disappearance of some 14C radioactivity from phosphatidylethanolamine and phosphatidylcholine, which might reflect activation of phospholipase A2. The activity of the phospholipase C could constitute the first step in the sequence of reactions that leads to the release of arachidonic acid.     

Association of changes in lysophosphatidylcholine metabolism and in microsomal membrane lipid composition to the pulmonary injury induced by oleic acid

Alterations in the lipid composition of lung microsomal membranes occur in oleic acid-induced respiratory distress. The marked decrease in the phosphatidylcholine/lysophosphatidylcholine molar ratio could be related with an altered metabolism of lysophosphatidylcholine in these membranes. Results revealed that the activity of phospholipase A increased whereas that of acyl-CoA:lysophosphatidylcholine acyltransferase decreased. Microsomal lysophospholipase activity remained unchanged. On the other hand, the microsomal enzyme system involved in the de novo synthesis of diacylglycerol was impaired, and cholinephosphotransferase activity was lowered. These changes in the activity of some membrane-bound enzymes were not caused by changes in the membrane lipid fluidity since lipid structural order parameter (SDPH) did not change and neither did the major factors on which the fluidity depends. The possible significance of microsomal lipid alterations in the pathogenesis of respiratory distress induced by oleic acid is discussed.     

Coenzyme A-mediated transacylation of sn-2 fatty acids from phosphatidylcholine in rat lung microsomes

Evidence was obtained for a CoA-dependent transfer of linoleate from rat lung microsomal phosphatidylcholine to lysophosphatidylethanolamine without the intervention of a Ca2+-requiring phospholipase A2 activity and ATP. To study this CoA-mediated transacylation process, microsomes were prepared in which the endogenous phosphatidylcholine was labeled by protein-catalyzed exchange with phosphatidylcholines containing labeled fatty acids in the sn-2-position. The apparent Km for CoA in the transfer of arachidonate from phosphatidylcholine to 1-acyllysophosphatidylethanolamine was 1.5 microM. At saturating lysophosphatidylethanolamine concentrations, the transacylation was linear with the amount of microsomal protein, i.e., a fixed percentage of the labeled fatty acid was transferred independent of the amount of microsomal protein. A maximal transfer of 12.2% for arachidonate and 2.0% for linoleate from the respective phosphatidylcholines to lysophosphatidylethanolamine was observed in 30 min. With 1-acyl-2-[1-14C]arachidonoylphosphatidylcholine as acyl donor, lysophosphatidylethanolamine was the best acceptor followed by lysophosphatidylglycerol and lysophosphatidylserine. Lysophosphatidate barely functioned as acceptor. These data provide further evidence for the widespread occurrence of CoA-mediated transacylation reactions. The arachidonate transacylation from phosphatidylcholine to other phospholipids in lung tissue may contribute to the low level of arachidonate in pulmonary phosphatidylcholine.     

Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.     

Regulation of phosphatidylcholine metabolism in mammalian hearts

Phosphatidylcholine is the major phospholipid in the mammalian heart. Over 90% of the cardiac phosphatidylcholine is synthesized via the CDP-choline pathway. The rate-limiting step of this pathway is catalyzed by CTP:phosphocholine cytidylyltransferase. Current evidence suggests that phosphatidylcholine biosynthesis in the heart is regulated by the availability of CTP and the modulation of cytidylyltransferase activity. Phosphatidylcholine is degraded mainly by the actions of phospholipase A1 and A2, with the formation of lysophosphatidylcholine. Lysophosphatidylcholine may be further deacylated by lysophospholipase or reacylated back into the parent phospholipid by the action of acyltransferase. The accumulation of lysophosphatidylcholine in the heart may be one of the biochemical factors for the production of cardiac arrhythmias.     

Characterization of phospholipase activities in chromaffin granule ghosts isolated from the bovine adrenal medulla

Highly purified chromaffin granule membranes contain high levels (100 nmol/mg protein) of long-chain free fatty acids (Husebye, E.S. and Flatmark, T. (1984) J. Biol. Chem. 259, 15272-15276), as well as lysophosphatidylcholine (268 nmol/mg protein) and lysophosphatidylethanolamine (92 nmol/mg protein). The release of saturated and unsaturated long-chain fatty acids from endogenous phospholipids was 38 and 28 nmol/mg protein per h, respectively, at 37 degrees C and pH 7.5 (alkaline pH optimum). p-Bromophenacyl bromide inhibited the release of palmitate and oleate by 88 and 65%, respectively. The deacylation of membrane phospholipids was not significantly affected by micromolar free Ca2+. Based on experiments with pancreatic phospholipase A2, stearate and arachidonate were found to be suitable markers for deacylation at the sn-1 and sn-2 positions, respectively. Experiments with exogenously added labeled phosphatidylcholines confirmed that chromaffin granule ghosts contain a phospholipase A2 activity (alkaline pH optimum). The preparations also revealed a phospholipase A1 activity (acid pH optimum). Finally, the ghosts contain a lysophospholipase activity (alkaline pH optimum), that accounts for the major part of the deacylation of membrane phospholipids, notably the release of saturated fatty acids (stearate and palmitate). It is unlikely that the high content of lysophospholipids is an artifact of the procedure by which the granule ghosts are isolated.     

Activities of some enzymes of phospholipid metabolism in cultured rat ventricular myocytes in normoxic and hypoxic conditions

Phospholipid catabolism is thought to be one of the critical events in membrane injury during heart ischemia. In this work, the enzymes involved in phospholipid metabolism were studied in purified cultured ventricular myocytes in normoxic and hypoxic conditions. Purified ventricular myocytes exhibited an alkaline phospholipase A activity which had sn-2 specificity and which was calcium dependent, and an acid phospholipase A activity with sn-1 specificity. These cells also exhibited lysophospholipase and acyl-CoA/lysophosphatidylcholine acyltransferase activities. Oxygen deprivation of the myocardial cells for 4 h resulted in a sharp reduction of both phospholipase A2 and A1 activities. The activities of the other lipolytic enzymes were unaffected by hypoxia. Although hypoxia resulted in a marked increase of lactate dehydrogenase leakage in the bathing fluid, no additional release of the lipolytic enzymes and mitochondrial enzyme was observed. However, we noted an important alkaline phospholipase A2 leakage during normoxia. It is suggested that ventricular myocytes, under hypoxia, tend to prevent phospholipid degradation by reducing their phospholipase A activities.     

Phospholipid-deacylating enzymes of rat stomach mucosa

1. Rat stomach mucosa exhibited three distinguishable phospholipid-deacylating enzyme activities: lysophospholipase, phospholipase A1 and phospholipase A2. 2. The lysophospholipase hydrolyzed 1-palmitoyl lysophosphatidylcholine to free fatty acid and glycerophosphorylcholine. This enzyme had an optimum pH of 8.0, was heat labile, did not require Ca2+ for maximum activity and was not inhibited by bile salts or buffers of high ionic strength. 3. Phospholipase A2 and phospholipase A1 deacylated dipalmitoyl phophatidylcholine to the corresponding lyso compound and free fatty acid. The specific activity of phospholipase A2 was 2--4-fold higher than that of phospholipase A1 under all the conditions tested. Both activities were enhanced 4--7.5-fold in the presence of bile salts at alkaline pH and 11-18-fold at acidic pH. 4. In the absence of bile salts, phospholipase A1 exhibited pH optima at 6.5 and 9.5 and phospholipase A2 at pH 6.5, 8.0 and 9.5. The pH optima for phospholipase A1 were shifted to pH 3.0, 6.0 and 9.0 in presence of sodium taurocholate; the activity was detected only at a single pH of 9.5 in the presence of sodium deoxycholate and at pH 10.0 in the presence of sodium glycocholate. Phospholipase A2 optimum activity was displayed at pH 3.0, 6.0 and 8.0 in presence of taurocholage, pH 7.5 and 9.0, in presence of glycocholate and only at pH 9.0 in presence of deoxycholate. 5. Ca2+ was essential for optimum activity of phospholipases A1 and A2. But phospholipase A1 lost complete activity in presence of 0.5 mM ethyleneglycolbis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) at pH 6.0, whereas phospholipase A2 lost only 50%. 6. Phospholipases A1 and A2 retained about 50% of their activities by heating at 75 degrees for 10 min. At 100 degrees, phospholipase A1 retained 22% of its activity, whereas phospholipase A2 retained only 7%.     

Influence of septic shock upon phosphatidylcholine remodeling mechanism in rat lung

Septic shock in rats lead to pulmonary disorders associated with alterations of phospholipid metabolism. The ratio between phosphatidylcholine and lysophosphatidylcholine is lowered both in lung tissue and in pulmonary surfactant because enzymes of phosphatidylcholine remodeling mechanism are distinctly affected by septic shock. Specific activity of phospholipase A2 is enhanced 5-fold while specific activities of lysolecithin acyltransferase and lysolecithin : lysolecithin acyltransferase are only slightly increased or remain unchanged. Beyond that, palmitic acid content of lung tissue phosphatidylcholine is significantly reduced and replaced mainly by arachidonic acid. The release of this fatty acid by action of phospholipase A2 may lead via intermediates to the generation of potent mediators such as prostaglandins, thromboxane or slow-reacting substance.     

Evidence for the reversibility of the acyl-CoA:lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver.

Acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine occurs in the microsomal preparations of developing safflower cotyledons. Evidence is presented to show that the acyl exchange is catalysed by the combined back and forward reactions of an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23). The back reaction of the enzyme was demonstrated by the stimulation of the acyl exchange with free CoA and by the observation that the added CoA was acylated with acyl groups from position 2 of sn-phosphatidylcholine. Re-acylation of the, endogenously produced, lysophosphatidylcholine with added acyl-CoA occurred with the same specificity as that observed with added palmitoyl lysophosphatidylcholine. A similar acyl exchange, catalysed by an acyl-CoA:lysophosphatidylcholine acyltransferase, occurred in microsomal preparations of rat liver. The enzyme from safflower had a high specificity for oleate and linoleate, whereas arachidonate was the preferred acyl group in the rat liver microsomal preparations. The rate of the back reaction was 3-5% and 0.2-0.4% of the forward reaction in the microsomal preparations of safflower and rat liver respectively. Previous observations, that the acyl exchange in safflower microsomal preparations was stimulated by bovine serum albumin and sn-glycerol 3-phosphate, can now be explained by the lowered acyl-CoA concentrations in the incubation mixture with albumin and in the increase in free CoA in the presence of sn-glycerol 3-phosphate (by rapid acylation of sn-glycerol 3-phosphate with acyl groups from acyl-CoA to yield phosphatidic acid). Bovine serum albumin and sn-glycerol 3-phosphate, therefore, shift the equilibrium in acyl-CoA:lysophosphatidylcholine acyltransferase-catalysed reactions towards the rate-limiting step in the acyl exchange process, namely the removal of acyl groups from phosphatidylcholine. The possible role of the acyl exchange in the transfer of acyl groups between complex lipids is discussed.     

Role of lamellar bodies in the biosynthesis of phosphatidylcholine in mouse lung.

1. A lamellar body-enriched fraction was isolated from whole lung homogenates of mouse lung and its contamination with microsomes, mitochondria, and cytosol protein assessed by marker enzyme analyses. 2. By measuring the activity of cholinephosphotransferase (EC 2.7.8.2) in varying amounts of microsomes in the presence and absence of a fixed quantity of lamellar bodies, it could be demonstrated unequivocally that lamellar bodies of mouse lung lack the capacity to synthesize phosphatidylcholine de novo. 3. A similar approach allowed the conclusion that lamellar bodies of mouse lung do not contain lysophosphatidylcholine acyltransferase (EC 2.3.1.23) and lysophosphatidylcholine:lysophosphatidylcholine acyltransferase (EC 2.3.1.--), enzymes which play a putative role in the formation of pulmonary 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine. The activities of these enzymes observed in lamellar body fractions could be attributed completely to contaminating microsomes and cytosol respectively. 4. Lamellar bodies contributed to the activity of microsomal lysophosphatidylcholine acyltransferase by a cooperative effect. The possible role of this cooperation in the biosynthesis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is discussed.     

Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.

We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.     

Acyltransferase activities in adult rat type II pneumocyte-derived subcellular fractions

Acyl-CoA: lysophosphatidylcholine, acyl-CoA: lysophosphatidylethanolamine, and lysophosphatidylcholine:lysophosphatidylcholine acyltransferases were investigated using subcellular fractions derived from adult rat type II pneumocytes in primary culture. Acyl-CoA:lysophospholipid acyltransferase activities were determined to be microsomal, while lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity was found to be cytosolic. Total palmitoyl CoA:lysophosphatidylcholine acyltransferase activity was 30-fold greater than lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity, indicating that the former enzyme is more important in the synthesis of dipalmitoyl phosphatidylcholine. Palmitoyl-CoA and oleoyl-CoA lysophosphatidylcholine acyltransferase activities were approximately equal under optimal substrate conditions. Specific activities of the enzyme using arachidoyl-CoA and arachidonoyl-CoA were 46% and 18%, respectively, of those with palmitoyl-CoA. Acyl-CoA:lysophosphatidylethanolamine acyltransferase showed a preference for palmitoyl-CoA as opposed to oleoyl-CoA under optimal conditions. However, when equimolar concentrations of either palmitoyl-CoA and oleoyl-CoA or palmitoyl-CoA and arachidoyl-CoA were assayed together, the relative utilization of the two substrates was found to be dependent on total acyl-CoA concentration. At higher concentrations, the incorporation of palmitoyl-CoA into phosphatidylcholine was less than other acyl-CoAs. However, at lower concentrations palmitoyl-CoA was utilized quite selectively. Whole lung microsomes did not show as marked a preference for palmitoyl-CoA as did type II pneumocyte microsomes under these same conditions. In similar experiments, low total acyl-CoA concentrations produced greater incorporation of oleoyl-CoA into phosphatidylethanolamine. For both enzymes total activity at the lowest concentrations used was at least 45% that at optimal conditions. This demonstrates that the type II pneumocyte acyltransferase system(s) can selectively utilize palmitoyl-CoA. No evidence for direct exchange of palmitoyl-CoA with 1-saturated-2-unsaturated phosphatidylcholine in subcellular fractions from type II pneumocytes was found.     

Modulation of phosphatidylcholine synthesis in vitro. Inhibition of diacylglycerol cholinephosphotransferase and lysophosphatidylcholine acyltransferase by centrophenoxine and neophenoxine

1,2-Diacyl-sn-glycerol : CDPcholine cholinephosphotransferase (EC 2.7.8.2) and acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) activities of rat liver microsomes can be inhibited by centrophenoxine (N,N-dimethylaminoethyl p-chlorophenoxyacetate). This inhibition is brought about by the intact centrophenoxine molecule rather than by the products of hydrolysis. A nonhydrolyzable ether analog of centrophenoxine was synthesized (neophenoxine; N,N-dimethylaminoethyl p-chlorophenoxyethyl ether) and proved most effective in inhibiting the two routes of phosphatidylcholine biosynthesis. While 50% inhibition of the cholinephosphotransferase was attained at 5 mM neophenoxine, 50% inhibition of the acyltransferase required 0.6 mM neophenoxine levels only. Inhibition of the cholinephosphotransferase (Ki approximately 1.5 mM) and the acyltransferase (Ki approximately 1 mM) by neophenoxine was shown to be noncompetitive. Other membrane-bound enzymes, such as glucose-6-phosphatase, monoacylglycerol lipase, alkaline phosphatase or phospholipase A2 were not affected by the inhibitors. Because of this specificity, and because of the high affinity of the microsomal membrane for such agents, centrophenoxine and neophenoxine should prove useful for controlling phosphatidylcholine synthesis and for modulating the phosphatidylcholine deacylation-reacylation cycle.     

Studies on the transverse localization of lysophospholipase in bovine liver microsomes using proteolytic enzymes.

1. Sonication of bovine liver microsomes completely solubilized the membrane-bound lysophospholipase II (EC 3.1.1.5). Co-chromatography with purified 125I-labelled lysophospholipase indicated that the enzyme was solubilized from microsomes in a lipid-free state. 2. In the presence of residual microsomal membranes, the solubilized lysophospholipase could only be partly degraded by trypsin (EC 3.4.21.4). Therefore, trypsin could not be used to study the transmembrane disposition of lysophospholipase in intact microsomes. 3. Chymotrypsin (EC 3.4.21.1) destroyed the solubilized lysophospholipase activity, even in the presence of residual microsomal membranes. 4. Lysophospholipase in intact microsomal vesicles was resistant to chymotrypsin digestion. 5. When microsomal vesicles were made leaky with lysophosphatidylcholine, chymotrypsin destroyed more than 95% of the lysophospholipase activity. 6. It is concluded from these experiments that at least the active center of lysophospholipase is located at the luminal side of the bovine liver microsomal membrane.     

R,R,R-alpha-tocopherol potentiates prostacyclin release in human endothelial cells. Evidence for structural specificity of the tocopherol molecule

Human umbilical vein endothelial cells (HUVEC) in culture synthesize prostacyclin (PGI2) as the predominant metabolite of arachidonic acid which is derived from the deacylation of phospholipids. Under basal-unstimulated condition, PGI2 release from HUVEC is extremely low; however, when endothelial monolayers were preincubated with the natural vitamin E (R,R,R-alpha-tocopherol), we found a dose-dependent potentiation of basal PGI2 release. When HUVEC were stimulated with arachidonate or ionophore A23187, there was a dose-dependent increase of PGI2 release in response to tocopherol enrichment. When HUVEC were labelled with [Me-3H]choline followed by A23187 stimulation, a significantly higher lysophosphatidylcholine was found in the tocopherol-enriched cells, suggesting a change in enzymes involved in phosphatidylcholine metabolism. Analysis of these enzymes revealed that phospholipase A2 activity was enhanced by tocopherol enrichment, whereas lysophospholipase and acyl-CoA acyltransferase were unaffected. To determine the specificity of the tocopherol molecule, different analogues were tested for their PGI2 potentiating activity. Results showed that the free hydroxyl group on the chromanol ring as well as the phytyl side-chain are absolutely required to stimulate PGI2 release, whereas, different methyl locations and substituents on the chromanol ring had no effect. These studies demonstrated that tocopherol potentiates basal PGI2 release in HUVEC and in contrast to its reported inhibitory role in rat platelets, myocardium and neutrophils, tocopherol stimulates phospholipase activity in HUVEC.     

Characterization of Lysophospholipid Metabolizing Enzymes in Human Brain

Abstract: Lysophospholipids are generated during the turnover and breakdown of membrane phospholipids. We have identified and partially characterized three enzymes involved in the metabolism of lysophospholipids in human brain, namely, lysophospholipase, lysophospholipid:acyl-CoA acyltransferase (acyltransferase), and lysophospholipid:lysophospholipid transacylase (transacylase). Each enzyme displayed comparable levels of activity in biopsied and autopsied human brain, although in all cases the activity was somewhat lower in human than that in rat brain. All three enzymes were localized predominantly in the particulate fraction, with lysophospholipase possessing the greatest activity followed by acyltransferase and transacylase. Lysophosphatidylcholine possessed a K_m in the micromolar range for lysophospholipase and transacylase, and in the millimolar range for acyltransferase, whereas arachidonyl-CoA displayed a K_m in the micromolar range for acyltransferase. The three enzymes differed in their pH optima, with lysophospholipase being most active at pH 8.0, transacylase at pH 7.5, and acyltransferase at pH 6.0. Both bromophenacyl bromide and N-ethylmaleimide inhibited lysophospholipase activity and, to a lesser extent, that of acyltransferase and transacylase. None of the enzyme activities were affected by the presence of dithiothreitol or EDTA, although particulate lysophospholipase was activated approximately two-fold by the addition of 5 mM MgCl₂ or CaCl₂ but not KCl. Transacylating activity was stimulated by CoA, the EC₅₀ of activation being 6.8 µM. Acyltransferase displayed an approximately threefold preference for arachidonyl-CoA over palmitoyl-CoA, whereas the acylation rate of different lysophospholipids was in the order lysophosphatidylinositol > 1-palmitoyl lysophosphatidylcholine > 1-oleoyl lysophosphatidylcholine ? lysophosphatidylserine > lysophosphatidylethanolamine. This, and the preference of human brain phospholipase A₂ for phosphatidylinositol, suggests that this phospholipid may possess a higher turnover rate than the other phospholipid classes examined. Human brain homogenates also possessed the ability to transfer fatty acid from lysophosphatidylcholine to lysophosphatidylethanolamine. In addition, we also present evidence that diacylglycerophospholipids can act as acyl donors for the transacylation of lysophospholipids. We have therefore demonstrated the presence of, and partially characterized, three enzymes that are involved in the metabolism of lysophospholipids in human brain. Our results suggest that lysophospholipase may be the major route by which lysophospholipids are removed from the cell membrane in human brain. However, all three enzymes likely play an important role in the remodeling of membrane composition and thereby contribute to the overall functioning of membrane-associated processes.     

Lipid metabolism by The gall-bladder. II. The in vitro conversion of lysophosphatidylcholine to phosphatidylcholine.

Lysophosphatidylcholine acyltransferase, which catalyzes the acylation of lysophosphatidylcholine with fatty acid coenzyme A to form phosphatidylcholine, was assayed in gall-bladder mucosa. In guinea pig gall-bladder the activity parallels that of the microsomal enzyme, glucose-6-phosphatase with 3--4-fold enrichment of the activity in the microsomes. Studies with saturated and unsaturated substrates demonstrated highest activity when oleoyl coenzyme A and palmitoyl lysophosphatidylcholine were used and the lowest activity when palmitoyl coenzyme A and palmitoyl lysophosphatidylcholine were used. This activity was demonstrated in the dog, rabbit, cat, calf and human gall-bladder mucosa; however, a wide variation in the amount was observed. Lysophospholipase, which catalyzes the hydrolysis of lysophosphatidylcholine to glycerophosphorylcholine and fatty acid, was also demonstrated in gall-bladder mucosa.     

Protein-mediated transbilayer movement of lysophosphatidylcholine in glycophorin-containing vesicles

1. Sonicated glycophorin-containing vesicles of dioleoyl phosphatidylcholine have been made. The outside-inside distributions of the lipid molecules in these vesicles was measured with NMR and was found to be comparable with that of protein-free vesicles. 2. The transbilayer distribution of palmitoyl lysophosphatidylcholine in these vesicles is such that they have a significantly higher content of the lyso-compound in the inner monolayer when compared with vesicles without glycophorin. 3. Lysophosphatidylcholine, added to pre-existing glycophorin-containing vesicles, is incorporated in the outer monolayer of these vesicles. Subsequently it is able to move to the inner monolayer with an estimated half time of about 1.5 h at 4 degrees C. This was measured with 13C-NMR using [N-13CH3]lysophosphatidylcholine. 4. Treatment of co-sonicated vesicles of phosphatidylcholine and lysophosphatidylcholine containing glycophorin with the enzyme lysophospholipase results in a complete degradation of the lyso-compound. A half time of transbilayer movement of lysophosphatidylcholine during this experiment was estimated to be about 1 h at 37 degrees C.