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.     

Calcium-independent phospholipase A2 in isolated rabbit ventricular myocytes

We characterized phospholipase A₂ (PLA₂) activity in isolated rabbit ventricular myocytes with respect to subcellular distribution, substrate specificity, and Ca²⁺ dependency. Membrane-associated PLA₂ was found to be an order of magnitude greater than cytosolic PLA₂. Ventricular myocyte PLA₂ activity was enhanced following protease-activated receptor stimulation with thrombin and was found to be largely Ca²⁺-independent and selective for phospholipid substrates containing arachidonic acid at the sn-2 position. Immunoblot analysis using an antibody to cytosolic Ca²⁺-independent PLA₂ from Chinese hamster ovary cells recognized a membrane-associated protein with a molecular mass of approximately 80 kDa; however, differences in pH optima, response to inhibitors, and substrate selectivity of membrane-associated and cytosolic PLA₂ activity suggest the presence of multiple Ca²⁺-independent PLA₂. Pretreatment with bromoenol lactone, a specific inhibitor of Ca²⁺-independent PLA₂, significantly attenuated membrane-associated and cytosolic PLA₂ in unstimulated and thrombin-stimulated myocytes. Pretreatment with methyl arachidonyl fluorophosphonate, mepacrine, or dibucaine had no significant effect on PLA₂ activity under all conditions tested. Ventricular myocyte PLA₂ activity was significantly inhibited by ATP, GTP, and their nonhydrolyzable analogs and was regulated by protein kinase C activity. These studies demonstrate the presence of one or more unique membrane-associated Ca²⁺-independent PLA₂ in isolated ventricular myocytes that exhibit a preference for phospholipids with arachidonate at the sn-2 position and that are activated by thrombin stimulation.     

1-O-alk-1′-enyl-2-acyl-glycerophosphoethanolamine content and molecular species composition in fish brain

Ethanolamine glycerophospholipids from the brains of both trout and cod comprised 36–38% of 1-O-alk-1′-enyl-2-acyl-glycerophosphoethanolamine (GPE) determined using two methods. In 1-O-alk-1′-enyl-2-acyl-GPE from trout brain, the main molecular species were 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1, which totalled 63.3%, while polyunsaturated fatty acid (PUFA) containing species totalled only 18.2%. 1-O-Alk-1′-enyl-2-acyl-GPE from cod brain was much more unsaturated with PUFA containing species totalling 52.6%, of which 18∶0a/20∶5n−3, 18∶1a/20∶5n−3 and 18∶1a/22∶6n−3 were predominant. In cod 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1 were the only other species present at over 5% each, totalling 31.8%. In both cod and trout, small amounts of species containing 22∶4n−6 were found. The results of this and earlier studies indicate that there is considerable specificity of composition at the level of molecular species between different lipid classes and subclasses. Molecular species of 1-O-alk-1′-enyl-2-acyl-GPE are abbreviated as follows:e.g., 16∶0a/18∶1 GPE is 1-O-hexadec-1′-enyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. The corresponding diacyl species, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, is abbreviated as 16∶0/18∶1.     

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.     

Molecular cloning,expression, and characterization of secretory phospholipase A<Subscript>2</Subscript> in tobacco

Phospholipase A₂ (PLA₂) activity was investigated in various tissues of tobacco (Nicotiana tabacum). PLA₂ activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA₂ activity in the flower appears to have originated from both Ca²⁺-dependent and-independent PLA₂. A cDNA clone for protein with homology to animal secretory PLA₂ (sPLA₂), denoted as Nt PLA₂, was isolated from the tobacco flower. The cDNA of Nt PLA₂ encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence for mature Nt PLA₂ contains 12 cysteines, a Ca²⁺ binding loop, and a catalytic domain that are commonly conserved in animal sPLA₂. The Nt PLA₂ mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA₂ was expressed as a fusion protein with thioredoxin in Escherichia coli. From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then the recombinant mature Nt PLA₂ was purified by ion exchange chromatography. It was discovered that the purified Nt PLA₂ essentially requires Ca²⁺, for the enzyme activity when the activity was determined using mixed-micellar phospholipid substrates with sodium cholate. The optimal activity of Nt PLA₂ was at pH 8–10 when PC was used as a substrate.     

Production of Structured Phosphatidylcholine with High Content of DHA/EPA by Immobilized Phospholipase A1-Catalyzed Transesterification

This paper presents the synthesis of structured phosphatidylcholine (PC) enriched with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) by transesterification of DHA/EPA-rich ethyl esters with PC using immobilized phospholipsase A₁ (PLA₁) in solvent-free medium. Firstly, liquid PLA₁ was immobilized on resin D380, and it was found that a pH of 5 and a support/PLA₁ ratio (w/v) of 1:3 were the best conditions for the adsorption. Secondly, the immobilized PLA₁ was used to catalyze transesterification of PC and DHA/EPA-rich ethyl esters. The maximal incorporation of DHA and EPA achieved was 30.7% for 24 h of reaction at 55 °C using a substrate mass ratio (PC/ethyl esters) of 1:6, an immobilized PLA₁ loading of 15% and water dosage of 1.25%. Then the reaction mixture was analyzed by ³¹P nuclear magnetic resonance (NMR). The composition of reaction product included 16.5% PC, 26.3% 2-diacyl-sn-glycero-3-lysophosphatidylcholine (1-LPC), 31.4% 1-diacyl-sn-glycero-3-lysophosphatidylcholine (2-LPC), and 25.8% sn-glycerol-3-phosphatidylcholine (GPC).     

Characterization of glycerophosphocholine phosphodiesterase activity and phosphatidylcholine biosynthesis in cultured retinal microcapillary pericytes. Effect of adenosine and endothelin-1

In pericytes from bovine retina, the enzyme glycerophosphocholine phosphodiesterase, catalyzing the hydrolysis of sn-glycero-3-phosphocholine to glycero-3-phosphate and choline, has been characterized with respect to pH optimum, metal ion dependence, K _m, inhibitors, and subcellular localization. In these cells, the natural substrate sn-glycero-3-phosphocholine was present at relatively high concentration (6.4±1.2 nmol/mg protein), and the EDTA-sensitive phosphodiesterase activity was also found to be markedly high (9.80±1.5 nmol/min/mg protein) compared to the estimated in liver and brain (1–3 nmol/min/mg protein) or in renal epithelial cell culture (0.27 nmol/min/mg protein). The reaction conditions were in general agreement with those found earlier in brain and other tissues. The majority of the enzyme specific activity was located in the plasma membrane, whereas a minor part was present in the microsomal fraction. The physiological significance of the high catabolic phosphodiesterase activity in these cells may be related to the tranfer, followed by deacylation, of lysophosphatidylcholine from the bloodstream to nervous tissue. In addition, capillary pericytes in culture were able to incorporate ³H-choline rapidly into choline-containing soluble phosphorylated intermediates and into phosphatidylcholine. To find a positive and negative effector on phosphatidylcholine formation, adenosine, an important intercellular mediator in the retina in response to alterations in oxygen delivery, and endothelin-1, a potent paracrine mediator present at the blood-brain and blood-retina barrier, were tested. The cells cultured for 1 or 24 h in a medium containing adenosine at concentrations of 10⁻⁶ and 10⁻⁴ M showed significant reduction in ³H-choline incorporation compared to control cultures, whereas endothelin-1, at a concentration of 10 and 100 nM, caused stimulation of phosphatidylcholine biosynthesis. These findings provide evidence that both agonists may modulate phosphatidylcholine metabolism in pericytes.     

Phospholipases A<Subscript>1</Subscript> from <Emphasis Type="Italic">Armillaria ostoyae</Emphasis> Provide Insight into the Substrate Recognition of α/β-Hydrolase Fold Enzymes

Four enzymes with phospholipase A₁ (PLA₁) activity were purified from the fruiting bodies of the basidiomycete Armillaria ostoyae. The enzymes (PLA₁-1, -2, -3 and -4) showed similar isoelectric points (4.3, 3.9, 4.0 and 4.0) and apparent molecular masses in the range of 35–47 kDa. Mass spectrometric analyses of proteolytic fragments revealed sequences homologous to α/β-hydrolase fold enzymes. The enzymes share one conserved region with fungal phospholipases B and the active site sequence with bacterial esterases and PLA₁s. PLA₁-1 cleaves phospholipids and lysophospholipids with an optimum activity at pH 5.3. In contrast, PLA₁-2, -3 and -4 are characterized by broad pH optima in the slightly acidic to neutral range and are additionally capable of hydrolyzing mono- and diglycerides as well as fatty acid methyl esters. All enzymes favor glycerol-based lipids with a single medium-sized fatty acid moiety in the sn-1 position but show reduced activity towards the corresponding 1,2-diacyl derivatives with bulky long-chain or inflexible saturated fatty acid moieties in the sn-2 position. The enzymes prefer zwitterionic phospholipid substrates and are unable to hydrolyze triglycerides. From the selectivity of these broad-spectrum α/β-hydrolase fold enzymes towards the different classes of their substrates a regiospecific steric hindrance and a head group recognition are concluded.     

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.     

Immobilization of Lipid Substrates: Application on Phospholipase A2 Determination

The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A₂ (PLA₂) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C₁₂‐NBD‐PtdCho, a fluorescent analogue of phosphatidylcholine, exhibited excellent stability for 3 months at 4 °C and was not desorbed in the aqueous reaction mixture during analysis. The limit of detection was 0.5 pmol FA (0.2 pg) and the linear part of the response curve extended from 1 up to 190 nmol FA/h/mL sample. The intra‐ and inter‐day relative standard deviations (%RSD), were ≤6 and ≤9 %, respectively. Statistical comparison with other fluorescent methods showed excellent correlation and agreement. Semiempirical calculations showed a fair amount of electrostatic interaction between the NBD‐labeled substrate and the crosslinked polyvinyl alcohol with the styryl pyridinium residues (PVA‐SbQ) material, from the plane of which, the sn‐2 acyl chain of the phospholipid stands out and is accessible by PLA₂. Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA₂. Mass spectrometry showed that only C₁₂‐NBD‐FA, the PLA₂ hydrolysis product, was detected in the reaction mixture, indicating that PLA₂ recognizes PVA‐SbQ/C₁₂‐NBD‐PtdCho as a surface to perform catalysis.     

The existence of a soluble plasmalogenase in guinea pig tissues

The distribution of plasmalogenase for the hydrolysis of 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) in the subcellular fractions of guinea pig tissues was examined. Plasmalogenase activity was found in high abundance in the cytosolic fractions of the brain and the heart. Assessment of microsomal marker enzyme activities in the cytosolic fraction revealed that plasmalogenase activity in the cytosol was not due to microsomal contaminations. The characteristics of the cytosolic plasmalogenase were very similar to the microsomal enzyme with respect to the pH profile of the reaction, the presence of divalent cations and K_m values for plasmenylethanolamine. However, the cytosolic enzyme was slightly less stable at 55°C than the microsomal enzyme. Cytosolic enzyme activity was eluted as a broad peak in Sepharose 6B chromatography with an average molecular weight of 250,000. Our results demonstrate that most of brain plasmalogenase activity is soluble which makes the brain cytosol an excellent source to initiate the purification of this enzyme.     

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.     

Platelet-activating factor acetylhydrolase activity in human tissues and blood cells

Human tissues, blood cells, and plasma have enzymes that catalyze the hydrolysis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The activities are not due to phospholipases A₂ that hydrolyze long chain acyl groups at thesn-2 position of glycerophospholipids, since they are calcium-independent and are specific for hydrolysis of short chain acyl groups. We examined the biochemical properties of these PAF acetylhydrolase activities (EC 3.1.1.47) in homogenates of human liver and spleen, in white blood cells (neutrophils and monocytes), and in erythrocytes. The data suggest that the plasma and intracellular PAF acetylhydrolase activities are likely due to different proteins. Second, the intracellular PAF acetylhydrolase activities in liver and spleen share several biochemical features that differentiate them from the activities in blood cells. Third, the activities in monocytes and neutrophils have properties that differentiate them from the activity present in human erythrocytes. Finally, the erythrocyte activity has unique properties that place it in a separate category of short chain acylhydrolases. In conclusion, there is a family of distinct enzymes that can be identified as PAF acetylhydrolases based on their calcium-independence and specificity for a short residue at thesn02 position of phospholipids. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Hydrocarbon chain distribution of ether phospholipids of the ascidianHalocynthia roretzi and the sea urchinStrongylocentrotus intermedius

The contents and compositions of the 1-O-alk-1′-enyl-2-acyl, 1-O-alkyl-2-acyl, and 1,2-diacyl glycerophospholipids in the muscle and viscera of the ascidianHalocynthia roretzi, and of the gonad of the sea urchinStrongylocentrotus intermedius, which are eaten to some extent in Alaska and in Asia, were analyzed by gas-liquid chromatography. 1-O-Alk-1′-enyl-2-acyl glycerophospholipids were found in all of the samples, accounting for 64.4–69.0% of the ethanolamine glycerophospholipid (EPL). By contrast, the levels of the 1-O-Alk-1′-enyl-2-acyl choline glycerophospholipids (CPL) were low (3.1–5.7%). CPL was rich in the 1-O-alkyl-2-acyl subclass amounting to 12.5–23.9% in the ascidian sample. The level of CPL in the sea urchin gonad was extremely high, amounting to 46.1%. The most prominent alkyl chains in thesn-1 position of CPL from the ascidian muscle were 16∶0 (44.6%), 18∶1 (26.5%), and 18∶0 (10.7%), and of CPL from the sea urchin gonad were 18∶0 (36.2%), 16∶0 (33.0%), and 18∶1 (17.8%). Unusually high levels of odd-numbered alkyl chains, e.g., 19∶0 andanteiso 17∶0, were detected in the CPL of all samples. The prominent alkenyl chains of EPL were 18∶0 (69.4%), 16∶0 (10.0%), and 18∶1 (8.54%) (not counting the vinyl double bond) for the sea urchin gonad. Relatively high levels of 20∶1 alkenyl chains were also present. The glycerolsn-2 positions contained high proportions of polyunsaturated fatty acids. Thus, 20∶5n-3 (43.6%) and 22∶6n-3 (20.1%) were most abundant in the alkylacyl CPL from the ascidian muscle and 20∶5n-3 (54.9%) and 20∶4n-6 (30.1%) in alkylacyl CPL from the sea urchin gonad. Despite a possible interconversion of the alkyl and alkenyl chains of each class of the ether phospholipids, they showed few features in common.     

Characterization of Secretory Phospholipase A<Subscript>2</Subscript> with Phospholipase A<Subscript>1</Subscript> Activity in Tobacco, <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> (L.)

A cDNA encoding protein with homology to plant secretory phospholipase A₂ (sPLA₂), denoted as Nt1 PLA₂, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA₂ has 12 cysteines, Ca²⁺ binding loop and catalytic site domain that are commonly conserved in plant sPLA₂s. The recombinant Nt1 PLA₂ was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA₂ could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²⁺ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA₂ mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.     

Effect of fortified milk on lyso‐platelet‐activating factor acetyltranferase and lipoprotein‐associated phospholipase A2 in hypercholesterolemic adults

Hypercholesterolemia is associated with subclinical inflammation, characterised by elevated proinflammatory mediators. Lyso‐platelet‐activating factor acetyltransferase (lyso‐PAF AT) and lipoprotein‐associated phospholipase A₂ (Lp‐PLA₂) are two key metabolic enzymes of platelet‐activating factor (PAF), a potent inflammatory lipid mediator. Little information is available concerning the efficacy of a dietary intervention on the metabolism of PAF. The objective of the study was to evaluate the effect of fortified milk on the activity of these enzymes. Forty‐three adults (mean age 49.8 ± 8.1 years) with body mass index <35 kg/m², and total cholesterol >200 but <310 mg/dL were randomised to two groups; (i) intervention group received 500 mL/day (two glasses) of a low‐fat milk fortified with phytosterols, linoleic and alpha linolenic acids, vitamin C, vitamin E, vitamin A, vitamin B6, vitamin B12, folic acid, magnesium and selenium (n = 22), and (ii) placebo group received 500 mL/day of a conventional low‐fat milk (n = 21) for 3 months. Outcome measures were the activities of lyso‐PAF AT from leukocytes and serum Lp‐PLA₂ determined with established methods. None of the activities changed significantly during the study in the intervention group, lyso‐PAF AT (95% confidence interval: ?1.7, 2.3 nmol/min/mg; p = 0.246), and Lp‐PLA₂ (?7.8, 5.8 nmol/min/mL, p = 0.591). No difference was observed between the two groups. In conclusion, daily intake of two glasses of phytosterols, antioxidants, linoleic and linolenic acids via fortified milk for three months had no effect on the activity of either lyso‐PAF AT or Lp‐PLA₂. Practical applications: Platelet‐activating factor (PAF) was the first intact phospholipid known to have messenger functions in which the signaling results from the molecule binding to specific receptors on the plasma membrane or other membranes of the cell. It has a number of pro‐inflammatory properties, and affects several critical points of atherogenesis including thrombosis, inflammation, and oxidation. Fortification of milk with nutrients that possess anti‐inflammatory properties and administration to adults with elevated blood cholesterol could provide a means to controlling inflammatory process through the synthesis and degradation of PAF in a population group at risk for cardiovascular morbidity and mortality.     

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.     

Phospholipases A2: structure and function

Phospholipases A₂ (PLA₂) are an example of peripheral membrane proteins that must first bind to the phospholipid interface to allow phospholipid hydrolysis to occur. The interfacial (membrane) binding step plays a crucial role in the biological function of the enzyme and membrane affinity may be determined both by the phospholipid composition of the membrane and the properties of the interfacial binding surface of the protein. There are now three major categories of these enzymes, secreted PLA₂ (sPLA₂), cytosolic PLA₂ (cPLA₂) and Ca²⁺‐independent PLA₂ (iPLA₂). The structure and function of each category is discussed highlighting how both membrane binding and phospholipid substrate specificity may contribute to the overall functions of these enzymes.     

PGE<Subscript>2</Subscript> Release from Tryptase-Stimulated Rabbit Ventricular Myocytes is Mediated by Calcium-Independent Phospholipase A<Subscript>2</Subscript>γ

Inflammation is associated with cardiovascular disease, including myocardial infarction, atherosclerosis, myocarditis and congestive heart failure. Mast cells have been implicated in inflammation, but their precise role in cardiac inflammation remains unclear. Mast cells contain a variety of pre-formed granule-associated mediators, including tryptase. We have previously demonstrated that the majority of the phospholipase A₂ (PLA₂) activity in isolated rabbit ventricular myocytes is membrane-associated, calcium-independent and selective for plasmalogen phospholipids. We hypothesized that tryptase stimulation of rabbit ventricular myocytes would increase iPLA₂ activity, leading to increased arachidonic acid and prostaglandin E₂ (PGE₂) release. Isolated rabbit ventricular myocytes were stimulated with tryptase and iPLA₂ activity, arachidonic acid and PGE₂ release were measured. Tryptase stimulation increased iPLA₂ activity after 5 min. Activation of iPLA₂ was accompanied by increased arachidonic acid and PGE₂ release in tryptase-stimulated myocytes. However no increase in platelet activating factor was observed with tryptase stimulation. To distinguish between different iPLA₂ isoforms in the myocardium, we pretreated ventricular myocytes with the (R)- and (S)-enantiomers of bromoenol lactone (BEL) to selectively inhibit iPLA₂γ and β respectively. Pretreatment with (R)-BEL resulted in complete inhibition of tryptase-stimulated iPLA₂ activity, arachidonic acid and PGE₂ release, suggesting the iPLA₂γ is the predominant myocardial isoform activated by tryptase. These studies demonstrate that PGE₂ release from tryptase stimulated rabbit ventricular myocytes is mediated primarily by iPLA₂γ.