Distributions of hydroperoxide positional isomers generated by oxidation of 1-palmitoyl-2-arachidonoyl-<Emphasis Type="Italic">sn</Emphasis>-glycero-3-phosphocholine in liposomes and in methanol solution

The differences in distribution of geometric isomers of unsaturated PC hydroperoxides generated by free radical oxidation were compared, as corresponding hydroxy analogs, in heterogeneous liposomes and in a homogeneous methanol solution by using HPLC with UV detection due to the presence of conjugated dienes. Identification of fractionated peak components was carried out by GC-MS. When the oxidation of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, PC(16∶0/18∶2), was initiated in liposomes by a hydrophilic azo radical initiator, and in a methanol solution by a hydrophobic azo radical initiator, there was no significant difference in the relative percentages of 1-palmitoyl-2-(9-hydroxy-trans-10,trans-12-octadecadienoyl)-sn-glycero-3-phosphocholine (9-t,t-OH PC) and 1-palmitoyl-2-(13-hydroxy-trans-9,trans-11-octadecadienoyl)-sn-glycero-3-phosphocholine (13-t,t-OH PC) between the PC oxidized in liposomes and in the methanol solution. For the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, PC(16∶0/20∶4), the relative percentage of 1-palmitoyl-2-(5-hydroxy-trans-6,cis-8,11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (5-OH PC) was significantly higher (P<0.01) than that of 1-palmitoyl-2-(15-hydroxy-cis-5,8,11,trans-13-eicosatetraenoyl)-sn-glycero-3-phosphocholine (15-OH PC) in liposomes. For the homogeneous methanol solution of PC(16∶0/20∶4), the relative percentage of 5-OH PC was close to that of 15-OH PC. For the PC(16∶0/20∶4) oxidized in bulk with added pentamethylchromanol, the individual amount of 15-OH PC, 1-palmitoyl-2-(11-hydroxy-cis-5,8trans-12,cis-14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (11-OH PC), 1-palmitoyl-2-(12-hydroxy-cis-5,8,trans-10,cis-14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (12-OH PC), 1-palmitoyl-2-(8-hydroxy-cis-5,trans-9,cis-11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (8-OH PC), 1-palmitoyl-2-(9-hydroxy-cis-5,trans-7,cis-11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (9-OH PC), and 5-OH PC were close to each other compared to the corresponding values in liposomes and in methanol solution. The results obtained by gel permeation chromatography of the PC liposomes containing hydrophilic 2,2′-azobis-2-amidinopropane) dihydrochloride (AAPH) suggest that the AAPH added to the liposomes of PC(16∶0/20∶4) was partitioned into the water phase and out of the hydrophobic region of the fatty acyl moieties of the PC. These results confirm that the distance that exists in the bis-allylic carbons of the unsaturated fatty acyl moieties of PC from the interface between the hydrophilic region of PC and the water phases played an important role in influencing hydrogen abstraction to form a symmetrical distribution of hydroperoxide isomers in both the heterogeneous liposomes and the homogeneous methanol solution.     

Biphasic action of platelet-activating factor on isolated guinea-pig ileum

1-0-Hexadecyl-2-0-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) at 10⁻¹⁰-10⁻⁹ M induced slow contraction of isolated guinea-pig ilcal muscles and the contraction persisted for a long time. At a higher concentration of 10⁻⁷ M, this phospholipid induced more rapid, but not greater, contraction. At higher concentrations (10⁻⁶-10⁻⁵ M), this phospholipid induced a biphasic response: rapid contraction followed by relaxation. At high concentrations, this compound inhibited acetylcholine-induced contractions. The stimulatory effect of this phospholipid was ca. 300 times that of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine, while its inhibitory potency on induced contraction was similar to those of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine and its lyso derivative. It was suggested that the differences in effects on contraction of different concentrations of 1-0-hexadecyl- and 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine were due to the dual effects of these compounds on the ileum: a strong stimulatory effect and a moderate inhibitory effect on contraction.     

Resolution of radiolabeled molecular species of phospholipid in human platelets: Effect of thrombin

Resolution of individual molecular species of human platelet 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with [³H]arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2-diacyl molecular species. Eighty percent of [³H]-arachidonic acid incorporated into 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine in resting platelets was equally distributed between 1-palmitoyl-2-arachidonoyl and 2-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, while 70% of the radiolabel in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine was found in 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1-acyl-2-[³H]arachidonoyl molecular species of 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-acyl-2-arachidonoyl-sn-glycero-3-ethanolamine. There was also a slight increase in 1-O-alkyl-2-[³H]arachidonoyl-sn-glycero-3-phosphocholine and a significant increase in 1-O-alk-1′-enyl-2-[³H]arachidonoyl-sn-glycero-3-phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl-containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin-stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate.     

Molecular species composition of phosphatidylcholines during the development of the avian embryo brain

A comparative approach has been used to investigate the molecular species composition of phosphatidylcholine (PC) and its age variation throughout several developmental stages of chick and duck embryo brains. The brain PC consist of 15 major molecular species which do not undergo appreciable variation in their relative abundance either during embryonic development or between equivalent stages of maturation in the 2 avian species. In fact, a highly invariable molecular architecture of PC is shown in the developing organ. Molecular species containing saturated or monounsaturated fatty acids were dominant in all stages of development of the avian embryo brain. Among these molecular species, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine accounted for 75–80% of the total PC.     

The importance of the steric configuration of lysophosphatidylcholine in the lymphatic transport of fat

The importance of the steric configuration of lysophosphatidylcholine in the lymphatic transport of fat was investigated in bile fistula rats. It was found that the feeding of 1-palmitoyl-sn-glycero-3-phosphocholine increased the lymphatic output of phosphatidyl choline and triacylglycerol, while the feeding of 3-palmitoyl-sn-glycero-1-phosphocholine had no effect. In intestinal microsomes of the bile fistula rats, it was found that the lysophosphatidylcholine acyltransferase was stereospecific in acylating the 1-acyl-sn-glycero-3-phosphocholine enantiomer. The significance of these findings is briefly discussed.     

Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase

We have examined the biosynthetic pathway of triacylglycerols containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[¹⁴C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[¹⁴C]rici-noleoyl-sn-glycero-3-phosphocholine, ¹⁴C-labeled 2-linoleoyl-phosphatidylcholine (PC), and ¹⁴C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A₂ strongly favored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversion to triacylglycerols was blocked. In the separate incubations of 2-[¹⁴C]ricinoleoyl-PC and [¹⁴C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC. Out results demonstrate the proposed pathway: 2-oleoyl-PC→2-ricinoleoyl-PC→ricinoleate →triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.     

Lyso platelet activating factor (LysoPAF) and its enantiomer. Total synthesis and carbon-13 NMR spectroscopy

Described is a reaction sequence for the total synthesis of lyso platelet activating factor (lysoPAF; 1-O-alkyl-sn-glycero-3-phosphocholine) and its enantiomer. The procedure is versatile and yields optically pure isomers of defined chain length. The synthesis is equally suited for the preparation of lysoPAF analogues and its enantiomers with unsaturation in the long aliphatic chain. First,rac-1(3)-O-alkylglycerol is prepared by alkylation ofrac-isopropylideneglycerol with alkyl methanesulfonate followed by acid-catalyzed removal of the ketal group. The primary hydroxy group of alkylglycerol is then protected by tritylation, the secondary hydroxy group is acylated, and the protective trityl group is removed under mild acidic conditions with boric acid on silicic acid, essentially without acyl migration. Condensation of the diradylglycerol with bromoethyl dichlorophosphate in diethyl ether, hydrolysis of the resulting chloride, and nucleophilic displacement of the bromine with trimethylamine givesrac-1-O-alkyl-2-acylglycero-3-phosphocholine in good overall yield. The racemic alkylacylglycerophosphocholine is finally treated with snake venom phospholipase A₂ (Ophiophagus hannah) which affords 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) of natural configuration in optically pure form. The “unnatural” 3-O-alkyl-2-O-acyl-sn-glycerol-1-phosphocholine enantiomer, which is not susceptible to phospholipase A₂ cleavage, gives 3-O-alkyl-sn-glycero-1-phosphocholine upon deacylation with methanolic sodium hydroxide. Homogeneity and structure of the intermediates and final products were ascertained by carbon-13 nuclear magnetic resonance spectroscopy on monomeric solutions.     

Biochemical characterization of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase in rat spleen microsomes

Acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) ultrasonic disruption in the presence of 25% glycerol from rat spleen microsomes. About 26% of the enzymatic activity was recovered in the 225,000×g supernatant by this treatment, although the specific activity was slightly decreased compared with the original microsomes. The solubilized enzyme was remarkably susceptible to various kinds of metal ions. Sulfhydryl reagents such as p-chloromercuribenzoate and N-ethyl-maleimide significantly inhibited the enzyme reaction, suggesting that the enzyme is an SH enzyme. Based on the sedimentation pattern in sucrose density centrifugation, the isoelectric point, the kinetic characteristics and the sensitivity to tryptic digestion of microsomes, it appears that acetyl-CoA:lyso-PAF acetyltransferase does not differ from the acetyltransferase responsible for the transfer of acetate from acetyl-CoA to 1-acyl-2-lyso-sn-glycero-3-phosphocholine.     

Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design

The thermoalkalophilic lipase from Bacillus thermocatenulatusBTL2 exhibits a low phospholipase activity (lecithin/tributyrinratio 0.03). A single round of random mutagenesis of the BTL2gene followed by screening of 6000 transformants on egg-yolkplates identified three variants with 10–12-fold increasedphospholipase activities, corresponding to lecithin/tributyrinratios of 0.16–0.36. All variants were specific for thesn-1 acyl ester bond of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.Mutations occurred predominantly in the N-terminal part of BTL2with regions surrounding the predicted helix     

Ether lipid content and fatty acid distribution in rabbit polymorphonuclear neutrophil phospholipids

This study was undertaken to determine if rabbit neutrophils contain sufficient ether-linked precursor for the synthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activatin factor) by a deacylation-reacylation pathway. The phospholipids from rabbit peritoneal polymorphonuclear neutrophils were purified and quantitated, and the choline-containing and ethanolamine-containing phosphoglycerides were analyzed for ether lipid content. Choline-containing phosphoglycerides (37%), ethanolamine-containing phosphoglycerides (30%), and sphingomyelin (28%) were the predominant phospholipid classes, with smaller amounts of phosphatidylserine (5%) and phosphatidylinositol (<1%). The choline-linked fraction contained high amounts of 1-O-alkyl-2-acyl-(46%) and 1,2-diacyl-sn-glycero-3-phosphocholine (54%), with a trace of the 1-O-alk-1′-enyl-2-acyl species. The ethanolamine-linked fraction contained high amounts of 1-O-alk-1′-enyl-2-acyl-(63%) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (34%), and a low quantity of the 1-O-alkyl-2-acyl species (3%). The predominant 1-O-alkyl ether chains found in thesn-1 position of the choline-linked fraction were 16∶0 (35%), 18∶0 (14%), 18∶1 (26%), 20∶0 (16%), and 22∶0 (9%). The major 1-O-alk-1′-enyl ether chains found in thesn-1 position of the ethanolamine-linked fraction were 14∶0 (13%), 16∶0 (44%), 18∶0 (27%), 18∶1 (12%) and 18∶2 (3%). The major acyl groups in thesn-1 position of 1,2-diacyl-sn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycero-3-phosphoethanolamine were 16∶0, 18∶0 and 18∶1. The most abundant acyl group in thesn-2 position of all classes of choline- and ethanolamine-linked phosphoglycerides was 18⩺2. Although this work does not define the biosynthetic pathway for platelet activating factor, it does show that there is ample precursor present to support its synthesis by a deacylation-reacylation pathway.     

Membrane Binding of Parkinson's Protein α-Synuclein: Effect of Phosphorylation at Positions 87 and 129 by the S to D Mutation Approach

Human α-synuclein, a protein relevant in the brain with so-far unknown function, plays an important role in Parkinson's disease. The phosphorylation state of αS was related to the disease, prompting interest in this process. The presumed physiological function and the disease action of αS involves membrane interaction. Here, we study the effect of phosphorylation at positions 87 and 129, mimicked by the mutations S87A, S129A (nonphosphorylated) and S87D, S129D (phosphorylated) on membrane binding. Local binding is detected by spin-label continuous-wave electron paramagnetic resonance. For S87A/D, six positions (27, 56, 63, 69, 76, and 90) are probed; and for S129A/D, three (27, 56, and 69). Binding to large unilamellar vesicles of 100 nm diameter of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in a 1 : 1 composition is not affected by the phosphorylation state of S129. For phosphorylation at S87, local unbinding of αS from the membrane is observed. We speculate that modulating the local membrane affinity by phosphorylation could tune the way αS interacts with different membranes; for example, tuning its membrane fusion activity.     

Influence of Lipid Saturation,Hydrophobic Length and Cholesterol on Double-Arginine-Containing Helical Peptides in Bilayer Membranes

Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW⁵LAL⁸LALALAL¹⁶ALW¹⁹LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific ²H-labeled alanine residues within the central sequence for detection by solid-state ²H NMR spectroscopy. The resulting pattern of [²H]Ala quadrupolar splitting (Δν_q) magnitudes indicates the core helix for R^8,16GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R^8,16GWALP23 helix adopts primarily a surface orientation. The inclusion of 10–20 mol % cholesterol in DOPC bilayers drives more of the R^8,16GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.     

Acylation of lyso platelet-activating factor by splenocytes of the rainbow trout,Oncorhyncus mykiss

In mammalian systems, platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, (PAF) is rapidly inactivated by a deacetylation/reacylation system that produces 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine which is highly enriched in arachidonic acid. There is some evidence that n−3 fatty acids may have an impact on this system in humans but the nature of this impact is unclear. In rainbow trout, n−3 fatty acids are known to be essential dietary components which are derived through the food chain. Substantial quantities of n−3 fatty acids are found in trout membrane phospholipids. We show here that in sharp contrast to mammalian cells, trout cells acylate lyso platelet-activating factor, alkyl-GPC, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine, (lyso-PAF) with a high degree of specificity for n−3 fatty acids. When [³H]lysoPAF was incubated with these cells, only three molecular species of alkylacylglycerophosphocholine were produced, and 92% contained n−3 fatty acids. Since isolated membranes yielded similar results, it appears that the acylation proceedsvia a coenzyme A-independent transacylase as found in mammalian systems.     

Synthesis of trideuteratedO-alkyl platelet activating factor and lyso derivatives

Racemic heavy isotope analogs of 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) and 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF) were prepared for use as internal standards to facilitate quantitative studies based on mass spectrometry. Starting from pentadencane-1,15-diol andrac-glycerol-1,2-acetonide, a convergent synthesis of 1-O-[16′-²H₃]hexadecyl and 1-O-[18′-²H₃]octadecylrac-glycero-3-phosphocholine and their acetyl derivatives is described. Three deuterium atoms were introduced at the terminal position of the 1-O-alkyl group by displacement of thep-toluensulfonyl group from 1-O-alkyl-15′-p-toluensulfonate and 1-O-alkyl-17′-p-toluensulfonate with [²H₃]-methylmagnesium iodide. The 1-O-alkyl-17′-p-toluensulfonate was obtained by reaction of the 1-O-alkyl-15′-p-toluensulfonate with allylmagnesium bromide, followed by reductive ozonolysis and treatment withp-toluenesulfonyl chloride. The hydroxyl group at C-2 was protected by a benzyl group and removed at a late stage in the synthesis. This provided the corresponding lysoderivatives or allowed preparation of racemic PAF by subsequent acetylation of the free hydroxy group. The phosphocholine moiety was introduced at glycerol C-3 by reaction with bromoethyldichlorophosphate and trimethylamine. The synthetic compounds were analyzed by FAB/MS and GC/NICIMS. They were shown to contain less than 0.6% protium impurity.     

1-O-alkyl-linked phosphoglycerides of human platelets: Distribution of arachidonate and other acyl residues in the ether-linked and diacyl species

In this study, the 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine content of human platelets was determined. The distribution of arachidonate among the 1,2-diacyl, 1-O-alkyl-2-acyl, and 1-O-alk-l′-enyl-2-acyl classes of choline- and ethanolamine-containing phosphoglycerides was also assessed. The major platelet phospholipids were choline-containing phosphoglycerides (38%), ethanolamine-containing phosphoglycerides (25%) and sphingomyelin (18%), with smaller amounts of phosphatidylserine (11%) and phosphatidylinositol (4%). In addition to the diacyl class, the choline-linked fraction was found to contain both 1-O-alkyl-2-acyl (10%) and 1-O-alk-l′-enyl-2-acyl (9%) species. The ethanolamine-linked fraction, on the other hand, had an elevated level of the 1-O-alk-l′-enyl-2-acyl (60%) species and a small amount of the 1-O-alkyl-2-acyl component (4%). The major fatty acyl residues found in all classes of the choline and ethanolamine phospholipids were 16∶0, 18∶0, (Δ⁹), 18∶2(n−6) and 20∶4(n−6). The 1-O-alk-l and 1-O-alk-l′-enyl fraction of the ethanolamine-linked phospholipids also contained substantial amounts of 22∶4(n−6), 22∶5(n−3) and 22∶6(n−3) acyl chains. Arachidonate comprised 44% of the acyl residues in thesn-2 position of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine. Corresponding values for the diacyl and 1-O-alk-l′-enyl-2-acyl species were 23% and 25%, respectively, based on all 20∶4(n−6) being linked to thesn-2 position of all classes. In the ethanolamine-linked phosphoglycerides, arachidonate constituted 60%, 20% and 68% of the acyl groups in thesn-2 position of the 1,2-diacyl, 1-O-alkyl-2-acyl and 1-O-alk-l′-enyl-2-acyl classes, respectively. The content of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine appears sufficient to support the synthesis of platelet activating factor by a deacylation-reacylation pathway in platelets. Our findings also demonstrate that human platelets contain a significant amount of 1-O-alkyl-2-arachidonyl-sn-glycero-3-phosphocholine that could possibly serve as a precursor of both platelet activating factor and bioactive arachidonate metabolites.     

Critical analysis of phospholipid hydrolyzing activities in ripening tomato fruits. Study by spectrofluorimetry and high-performance liquid chromatography

Using the spectrofluorimetric method described by Wittenaueret al. [Wittenauer, L.A., Shirai, K., Jackson, R.L., and Johnson, J.D. (1984)Biochem. Biophys. Res. Commun. 118, 894–901] for phospholipase A₂ (PLA₂) measurement, we have detected a phospholipase activity in Ailsa Craig and in mutantrin tomatoes at their normal harvest time (mature green stage). This activity in Ailsa Craig tomatoes increased at the beginning of fruit ripening (green-orange stage) and then decreased slowly. The decrease in activity, however, was greater when ripening occurred after tomato picking at normal harvest time than when ripening occurred on tomato plants. This phospholipase activity was always higher inrin tomatoes than in normal ones. Thin-layer chromatography of compounds obtained after incubation of tomato extract demonstrated a decrease in the substrate 1-acyl-2-{6[(7-nitro-2,1,3, benzoxadiazol-4-yl)amino]-caproyl}-sn-glycero-3-phosphocholine (C₆-NBD-PC), and an increase in one product (NBD-aminohexanoic acid), but failed to detect the second product (1-acyl-sn-glycero-3-phosphocholine). We, therefore, developed a new one-step method for separation and quantification of a mixture of phospholipids and other lipids, using straight-phase-high-performance liquid chromatography with light-scattering detection. This method detected another fatty acid-releasing activity in enzyme extract from green-orange tomatoes. This lipolytic enzyme (or family of enzymes) slowly produced free fatty acids when 1-oleoyl-sn-glycero-3-phosphocholine was added as substrate. The production of fatty acids was stoichiometric and more rapid when 1-oleoyl-sn-glycero-3-phosphate and 1-oleoyl-sn-glycerol were used as substrates. On the other hand, the same tomato extract was unable to hydrolyze 1,2-dioleoyl-sn-glycero-3-phosphate and 1,2-dioleoyl-sn-glycerol. Crude tomato extract exhibited lipid acyl hydrolase activity according to the definition of Galliard [Galliard, T. (1979), inAdvances in the Biochemistry and Physiology of Plant Lipids (Appelqvist, L.A., and Liljenberg, C. eds.), pp. 121–132, Elsevier, Amsterdam]. But in order to demonstrate whether tomato extract contains PLA₂ activity and/or lysophospholipase activity, further work on purified tomato extract will be necessary.     

1-O-alk-1′-enyl-2-acyl and 1-O-alkyl-2-acyl glycerophospholipids in white muscle of bonitoEuthynnus pelamis (Linnaeus)

The existence of ether-linked phospholipids, including 1-O-alk-1′-enyl-2-acyl and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines and ethanolamines in bonitoEuthynnus pelamis (Linnaeus) white muscle, was investigated by gas chromatography and gas chromatography-mass spectrometry. Chemical ionization (iso-butane) mass spectrometry of trimethylsilyl ethers derived from the corresponding ether-linked glycerophospholipids proved effective not only for determining molecular weights but also for structural identification based on the ions [M−R]⁺, [M−RO]⁺ and [M+1]⁺. 1-O-Alk-1′-enyl-2-acyl-sn-glycero-3-phosphocholine and ethanolamine accounted for 3.0–6.0% and 3.6–7.6% of the total glycerophospholipids, respectively. 1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine and ethanolamine were also determined for one fish and accounted for 1.4% and 0.6% of the total glycerophospholipids, respectively. The predominant long chains in thesn-1 position of the glycerol moieties were 16∶0, 18∶0 and 18∶1 in the case of the alkenylacyl and alkylacyl components. Fatty acid distribution of individual glycerophospholipids was also determined.     

Purification of phospholipids by preparative high pressure liquid chromatography

A method is described for the purification of a number of phospholipids by preparative high performance liquid chromatography (HPLC). Purification of digalactosyl-diglyceride from spinach and egg phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine from its reaction mixture have been resolved. The lipid separation is performed on a polygosil column and the individual compounds are monitored directly by refractive index detection. Chloroform/methanol mixtures are used as eluent systems, providing a wide polarity range to separate the classes of lipids. The developed equipment can be used for columns between 10 and 50 cm long and 4 and 50 mm inner diameter. The flow rate could be varied between 1 and 100 ml/min and applied pressures between 10 and 450 bars.     

Stereospecific synthesis of antitumor active thioether PAF analogs

A novel stereospecific synthesis of antitumor active thioether analogs of platelet-activating factor (PAF) is reported. The synthesis is based upon: i) the use ofD-serine to provide the chiral center for the construction of the optically active phospholipid molecule; ii) development of thesn-1-thioalkyl function via thioacetate displacement of methanesulfonate-activated primary hydroxyl group followed by alkylation of thesn-1-thiolate function; and iii) introduction of the phosphocholine moiety through the 2-chloro-2-oxo-1,3,2-dioxaphospholane/trimethylamine sequence. The entire scheme relies on the use of a single protecting group. The synthetic thioether phospholipid 1-S-hexadecyl-2-N-acetamidodeoxy-sn-glycero-3-phosphocholine has been shown to be a potent antitumor active phospholipid, exhibiting tumor cytotoxicity against a lymphoblastoid lymphoma (Li-A) cell line and a malignant histiocytic (DHL-4) cell line of human origin at the same level of potency as ET-18-OMe and 1-O-octadecyl-2-N-acetamidodeoxy-sn-glycero-3-phosphocholine. The synthetic method described has a great deal of flexibility, providing a convenient general route to a wide range of thioether PAF analogs. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Synthesis of the dipalmitoyl species of diacyl glycerophosphocholine by rabbit alveolar macrophages

The distribution of radioactivity among the molecular species of diacyl glycerophosphocholine of rabbit alveolar macrophages was determined after incubation with [³H]glycerol and 1-[¹⁴C]palmitoyl-sn-glycero-3-phosphocholine. The highest percentage of radioactivity of [³H]glycerol was found in the dipalmitoyl species (35% of the total) followed by the 1-palmitoyl-2-linoleoyl (23.6%) and 1-stearoyl-2-linoleoyl plus 1-palmitoyl-2-oleoyl species (19.7%) during the first 30 min incubation. The radioactivity of the dipalmitoyl species reached a maximum at 120 min incubation and decreased thereafter, although the radioactivities of other molecular species still increased. In contrast to the [³H]glycerol labeling, only 4% of the total radioactivity in diacyl glycerophosphocholine derived from 1-[¹⁴C]palmitoyl glycerophosphocholine was found in the dipalmitoyl species; 80% of the radioactivity was located in the 1-palmitoyl-2-arachidonoyl species at 10 min incubation. The present results indicate that the dipalmitoyl species of diacyl glycerophosphocholine are synthesized predominantly via a de novo pathway and not the deacylation-reacylation pathway in rabbit alveolar macrophages.