Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7

The lipid mediators, platelet activating factor (PAF) and the eicosanoids, can be coordinately produced from the common phospholipid precursor, 1-O-alkyl-2-arachidonoylglycerophosphocholine (1-O-alkyl-2-arachidonoyl-GPC), through the initial action of a phospholipase A2 that cleaves arachidonic acid from the sn-2 position. The mouse macrophage cell line RAW 264.7, which was used as a model macrophage system to study the arachidonoyl-hydrolyzing phospholipase A2 enzyme(s), could be induced to release arachidonic acid in response to inflammatory stimuli. A phospholipase A2 that hydrolyzed 1-O-hexadecyl-2-[3H]arachidonoyl-GPC was identified in the cytosolic fraction of these macrophages. This phospholipase activity was optimal at pH 8 and dependent on calcium. Enzyme activity could be stimulated 3-fold by heparin, suggesting the presence of phospholipase inhibitory proteins in the macrophage cytosol. Compared to 1-alkyl-2-arachidonoyl-GPC, the enzyme hydrolyzed 1-acyl-2-arachidonoylglycerophosphoethanolamine (1-acyl-2-arachidonoyl-GPE) with similar activity but showed slightly greater activity against 1-acyl-2-arachidonoyl-GPC, suggesting no specificity for the sn-1 linkage or the phospholipid base group. Although comparable activity against 1-acyl-2-arachidonoylglycerophosphoinositol (1-acyl-2-arachidonoyl-GPI) could be achieved, the enzyme exhibited much lower affinity for the inositol-containing substrate. The enzyme did, however, show apparent specificity for arachidonic acid at the sn-2 position, since much lower activity was observed against choline-containing substrates with either linoleic or oleic acids at the sn-2 position. The cytosolic phospholipase A2 was purified by first precipitating the enzyme with ammonium sulfate followed by chromatography over Sephadex G150, where the phospholipase A2 eluted between molecular weight markers of 67,000 and 150,000. The active peak was then chromatographed over DEAE-cellulose, phenyl-Sepharose, Q-Sepharose, Sephadex G150 and finally hydroxylapatite. The purification scheme has resulted in over a 1000-fold increase in specific activity (2 mumol/min per mg protein). Under non-reducing conditions, a major band on SDS-polyacrylamide gels at 70 kDa was observed, which shifted to a lower molecular weight, 60,000, under reducing conditions. The properties of the purified enzyme including the specificity for sn-2-arachidonoyl-containing phospholipids was similar to that observed for the crude enzyme. The results demonstrate the presence of a phospholipase A2 in the macrophage cell line. RAW 264.7, that preferentially hydrolyzes arachidonoyl-containing phospholipid substrates.     

Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase activity

FtsY, the Escherichia coli homologue of the eukaryotic signal recognition particle (SRP) receptor alpha-subunit, is located in both the cytoplasm and inner membrane. It has been proposed that FtsY has a direct targeting function, but the mechanism of its association with the membrane is unclear. FtsY is composed of two hydrophilic domains: a highly charged N-terminal domain (the A-domain) and a C-terminal GTP-binding domain (the NG-domain). FtsY does not contain any hydrophobic sequence that might explain its affinity for the inner membrane, and a membrane-anchoring protein has not been detected. In this study, we provide evidence that FtsY interacts directly with E.coli phospholipids, with a preference for anionic phospholipids. The interaction involves at least two lipid-binding sites, one of which is present in the NG-domain. Lipid association induced a conformational change in FtsY and greatly enhanced its GTPase activity. We propose that lipid binding of FtsY is important for the regulation of SRP-mediated protein targeting.     

Kinetic properties of a high molecular mass arachidonoyl-hydrolyzing phospholipase A2 that exhibits lysophospholipase activity.

The first step in the production of eicosanoids and platelet-activating factor is the hydrolysis of arachidonic acid from membrane phospholipid by phospholipase A2. We previously purified from the macrophage cell line RAW 264.7 an intracellular phospholipase A2 that preferentially hydrolyzes sn-2-arachidonic acid. The enzyme exhibits a molecular mass of 100 kDa and an isoelectric point of 5.6. When assayed for other activities, the phospholipase A2 was found to exhibit lysophospholipase activity against palmitoyllysoglycerophosphocholine, and both activities copurified to a single band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. An antibody against the macrophage enzyme was found to quantitatively immunoprecipitate both phospholipase A2 and lysophospholipase activities from a crude cytosolic fraction. When the immunoprecipitated material was analyzed on immunoblots, a single band at 100 kDa was evident, further suggesting that a single protein possessed both enzyme activities. When assayed as a function of palmitoyllysoglycerophosphocholine concentration and plotted as a double-reciprocal plot, two different slopes were apparent, corresponding to concentrations above and below the critical micellar concentration (7 microM) of the substrate. Above the critical micellar concentration, lysophospholipase exhibited an apparent Km of 25 microM and a Vmax of 1.5 mumol/min/mg. Calcium was not required for lysophospholipase activity, in contrast to phospholipase A2 activity. The enzyme, when assayed as either a phospholipase A2 or lysophospholipase, exhibited nonlinear kinetics beyond 1-2 min despite low substrate conversion. Readdition to more substrate after the activity plateaued did not result in further enzyme activity, ruling out substrate depletion. Readdition of enzyme, however, resulted in another burst of enzyme activity. The results are not consistent with product inhibition, but suggest that the enzyme may be subject to inactivation during catalysis.     

A novel phosphatidylglycerol-selective phospholipase A2 from macrophages

In our recent studies on the synthesis of bis(monoacylglycero)phosphate (BMP), we postulated that the first step involved a PLA2 that cleaved the 2-acyl group from phosphatidylglycerol (PG). In the present study, a novel lysosomal PLA2 was partially purified and characterized from RAW 264.7, macrophage like cells. Cells were homogenized and delipidated, and the PLA2 activity in the soluble fraction was purified by Sephacryl S100 and DEAE Sephacel. Further purification was performed using Con-A Sepharose, Phenyl Sepharose, DEAE Sephacel, and Superdex 75 FPLC. The enzyme at this stage of purification showed a dominant band around 45 kDa plus several minor bands on SDS-PAGE. The molecular mass determined by Superdex 75 column FPLC was about 45 kDa. The highly purified fraction hydrolyzed at the sn-1 position, implying that this PLA2 also has some intrinsic PLA1 activity. This enzyme preferentially hydrolyzed PG, has an acidic pH optima, and does not require divalent metal ions. Comparison using PG with various acyl chains on the sn-2 position showed that oleate and linoleate were preferred relative to arachidonate. MAFP, a known cytosolic PLA2 inhibitor, strongly inhibited this PLA2 activity. MJ33, AACOCF3, DENP, and Amiodarone also gave moderate inhibition. The characteristics of this enzyme showed this to be a new type of PLA, and the overwhelming preference for PG as substrate suggests its physiological role is in the biosynthesis of BMP.     

Regulatory aspects of mitochondrial phospholipase A2 from rat liver: effects of proteins, phospholipids and calcium ions

This paper deals with the search for specific inhibitors or activators of the mitochondrial phospholipase A2. Convincing evidence for the existence of proteins in the mitochondrial or cytosolic fraction that function as specific regulators of this enzyme was not obtained. The enzymatic activity appeared to be inhibited at low substrate concentrations by lipocortin isolated from human monocytes. However, at higher substrate concentrations, the inhibition disappeared, suggesting either that lipocortin sequestered the phospholipid substrate or that the putative inactive complex of enzyme and lipocortin dissociated in the presence of excess phospholipids. The hydrolysis of the neutral phospholipid phosphatidylethanolamine was stimulated by the presence of cardiolipin and phosphatidylglycerol. It is unlikely that this is caused merely by the negative charge of these phospholipids, since other negatively charged phospholipids did not show this effect. Using a phospholipid extract from mitochondria as substrate, the enzymatic activity as a function of the Ca2+ concentration was determined. Only one enzyme activity plateau was observed. The calculated KCa2+ value of 0.05 mM suggests that the mitochondrial phospholipase A2 could be regulated strictly by the modulation of the free Ca2+ concentration in vivo. The two activity plateaus observed previously upon variation of the Ca2+ concentration using phosphatidylethanolamine as substrate could be explained by a Ca2+-induced transition of the phospholipid structure.     

Corticosteroids stimulate an increase in phospholipase A2 inhibitor in human serum

A corticosteroid induced increase in a circulating inhibitor of serum phospholipase A2 activity is described. Inhibitor activity was found to be normally present in serum in agreement with the findings of other workers, and this activity was significantly increased by either acute or chronic administration of corticosteroids. The possible relation of this inhibitor to the known inhibitory effects of lipocortin and sphingomyelin on phospholipase A2 activity is briefly discussed.     

Molecular basis of phospholipase A2 activity toward phospholipids with sn-1 substitutions

We studied secretory phospholipase A₂ type IIA (sPLA₂) activity toward phospholipids that are derivatized in the sn-1 position of the glycerol backbone. We explored what type of side group (small versus bulky groups, hydrophobic versus polar groups) can be introduced at the sn-1 position of the glycerol backbone of glycerophospholipids and at the same time be hydrolyzed by sPLA₂. The biophysical characterization revealed that the modified phospholipids can form multilamellar vesicles, and several of the synthesized sn-1 functionalized phospholipids were hydrolyzed by sPLA₂. Molecular dynamics simulations provided detailed insight on an atomic level that can explain the observed sPLA₂ activity toward the different phospholipid analogs. The simulations revealed that, depending on the nature of the side chain located at the sn-1 position, the group may interfere with an incoming water molecule that acts as the nucleophile in the enzymatic reaction. The simulation results are in agreement with the experimentally observed sPLA₂ activity toward the different phospholipid analogs.     

Modulation of purified phospholipase A2 activity from human platelets by calcium and indomethacin.

A membrane bound phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human platelets has been purified 3500-fold, and partially characterized. Phospholipase A2 activity was assayed using [1(-14)C] oleate-labeled Escherichia coli or sonicated dispersions of synthetic phospholipids. The 2-acyl specificity of the phospholipase activity was confirmed using phosphatidylethanolamine labeled in the C-1 position as substrate. The purified enzyme was maximally active between pH 8.0 and 10.5, and had an absolute requirement for low concentrations of Ca2+. Indomethacin, but not aspirin, inhibited phospholipase A2 activity.     

The activation of phospholipase C from Clostridium Welchii by quinine: an absolute requirement for calcium ions.

Quinine activates the hydrolysis of phosphatidyl choline suspensions by phospholipase C (E.C. 3.1.4.3) obtained from Clostridium welchii. Low levels of calcium are an absolute requirement for this activation: Mg2+, Ba2+, Sr2+, and Zn2+ are ineffective. The induction period, or lag phase for this enzyme is dependent upon both calcium concentration and substrate interfacial surface area. At low concentrations (less then 50 muM) calcium ions affect the induction period but not the maximal rate of hydrolysis, whereas guinine predominantly affects the rate of hydrolysis by alterations in the surface charge carried by the substrate.     

A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7

Arachidonoyl-hydrolyzing phospholipase A2 plays a central role in providing substrate for the synthesis of the potent lipid mediators of inflammation, the eicosanoids, and platelet-activating factor. Although Ca2+ is required for arachidonic acid release in vivo and most phospholipase A2 enzymes require Ca2+ for activity in vitro, the role of Ca2+ in phospholipase A2 activation is not understood. We have found that an arachidonoyl-hydrolyzing phospholipase A2 from the macrophage-like cell line, RAW 264.7, exhibits Ca2(+)-dependent association with membrane. The intracellular distribution of the enzyme was studied as a function of the Ca2+ concentration present in homogenization buffer. The enzyme was found almost completely in the 100,000 x g soluble fraction when cells were homogenized in the presence of Ca2+ chelators and there was a slight decrease in soluble fraction activity when cells were homogenized at the level of Ca2+ in an unstimulated cell (80 nM). When cells were homogenized at Ca2+ concentrations expected in stimulated cells (230-450 nM), 60-70% of the phospholipase A2 activity was lost from the soluble fraction and became associated with the particulate fraction in a manner that was partly reversible with EGTA. Membrane-associated phospholipase A2 activity was demonstrated by [3H]arachidonic acid release both from exogenous liposomes and from radiolabeled membranes. With radiolabeled particulate fraction as substrate, this enzyme hydrolyzed arachidonic acid but not oleic acid from membrane phospholipid, and [3H]arachidonic acid was derived from phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol/phosphatidylserine. We suggest a mechanism in which the activity of phospholipase A2 is regulated by Ca2+: in an unstimulated cell phospholipase A2 is found in the cytosol; upon receptor ligation the cytosolic Ca2+ concentration increases, and the enzyme becomes membrane-associated which facilitates arachidonic acid hydrolysis.     

Monosodium urate crystals stimulate phospholipase A2 enzyme activities and the synthesis of a phospholipase A2-activating protein

Eicosanoids are important mediators of the inflammatory response to monosodium urate crystals (MSUC) that results in gout. Phospholipase enzymes cleave fatty acids from membrane phospholipids, and this is thought to be the rate-limiting step in eicosanoid production. To understand better the mechanism of eicosanoid production in this disease, we stimulated human peripheral blood neutrophils and monocytes with MSUC and measured phospholipase enzyme activities. MSUC stimulated both intracellular and secretory phospholipase A2 enzyme activities in a time and concentration-dependent manner. Specificity was observed, as phospholipase C activities were not affected. Pretreatment with colchicine, but not aspirin, indomethacin, allopurinol, or islet activating protein, abrogated the enhanced phospholipase A2 activities. We have recently isolated and characterized a phospholipase A2 activating protein termed PLAP from synovial fluid from patients with rheumatoid arthritis, and from murine and bovine cell lines. PLAP was detected in gouty synovial fluid by immunodot blotting and ELISA assays and expressed the same characteristics as PLAP identified from other sources. To examine the role of PLAP in MSUC-induced phospholipase A2 stimulation, we treated cells with MSUC and observed an increase in immunoreactive PLAP. This response also could be blunted by colchicine, but not other drugs. Both phospholipase A2 and PLAP induced production by human monocytes of PGE2 and leukotriene B4 by neutrophils. These findings suggest that phospholipase A2 activation in response to MSUC requires an intact microtubule structure, and that phospholipase A2 and PLAP may be important modulators of at least a portion of the gouty inflammatory response.     

A phospholipase B from Pseudomonas aeruginosa with activity towards endogenous phospholipids affects biofilm assembly

Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and biofilm-mediated multidrug resistance. It produces and secretes various toxins with hydrolytic activities including phospholipases. However, the function of intracellular phospholipases for bacterial virulence has still not been established. Here, we demonstrate that the hypothetical gene pa2927 of P. aeruginosa encodes a novel phospholipase B named PaPlaB. At reaction equilibrium, PaPlaB purified from detergent-solubilized membranes of E. coli released fatty acids (FAs) from sn-1 and sn-2 positions of phospholipids at the molar ratio of 51:49. PaPlaB in vitro hydrolyzed P. aeruginosa phospholipids reconstituted in detergent micelles and phospholipids reconstituted in vesicles. Cellular localization studies indicate that PaPlaB is a cell-bound PLA of P. aeruginosa and that it is peripherally bound to both membranes in E. coli, yet the active form was predominantly associated with the cytoplasmic membrane of E. coli. Decreasing the concentration of purified and detergent-stabilized PaPlaB leads to increased enzymatic activity, and at the same time triggers oligomer dissociation. We showed that the free FA profile, biofilm amount and architecture of the wild type and ΔplaB differ. However, it remains to be established how the PLB activity of PaPlaB is regulated by homooligomerisation and how it relates to the phenotype of the P. aeruginosa ΔplaB. This novel putative virulence factor contributes to our understanding of phospholipid degrading enzymes and might provide a target for new therapeutics against P. aeruginosa biofilms.     

Synergistic effect of magnesium and calcium ions in the activation of phospholipase A2 of liver macrophages

In cell-free extracts of rat liver macrophages (Kupffer cells) phospholipase A2 was found to be strongly activated at free Ca2+ concentrations from 100 nM to 1 microM in the presence of 4 mM free Mg2+. This is within the range of intracellular free Ca2+ reported for basal and various stimulated conditions, respectively. Ca2+ alone increased phospholipase A2 activity at high Ca2+ concentrations (1 mM) whereas Mg2+ alone had only little stimulatory effect. Calmodulin does not seem to participate in the regulation of phospholipase A2 although it relieved the inhibition of phospholipase A2 activity by calmodulin antagonists.     

Implication of lipoprotein associated phospholipase A2 activity in oxLDL uptake by macrophages

Recognition and uptake of oxidized LDL (oxLDL) by scavenger receptors of macrophages and foam cell formation are mediated by the oxidatively modified apolipoprotein B (ApoB) and lipid moiety of oxLDL. A great amount of oxidized phosphatidylcholine (oxPC) of oxLDL is hydrolyzed at the sn-2 position by lipoprotein associated phospholipase A₂ (Lp-PLA₂) to lysophosphatidylcholine and small oxidation products. This study examines the involvement of Lp-PLA₂ in the uptake of oxLDL by mouse peritoneal macrophages. LDL with intact Lp-PLA₂ activity [LDL (+)] and LDL with completely inhibited Lp-PLA₂ activity [LDL (-)] were subjected to oxidation with 5 μM CuSO₄ for 6 h [moderately oxLDL (MoxLDL)], or 24 h [heavily oxLDL (HoxLDL)] and peritoneal macrophages were incubated with these preparations. The uptake of MoxLDL(-) was about 30% increased compared with that of MoxLDL(+), and HoxLDL(-) uptake was about 20% increased compared with that of HoxLDL(+). Inhibition of Lp-PLA₂ activity had no effect on the uptake of ApoB-liposomes conjugates with ApoB isolated from MoxLDL(-), MoxLDL(+), HoxLDL(-), and HoxLDL(+). Liposomes prepared from the lipid extract of MoxLDL(-), MoxLDL(+), HoxLDL(-), and HoxLDL(+) exhibited a similar pattern to that observed in the uptake of the corresponding intact lipoproteins. This study suggests that the progressive inactivation of Lp-PLA₂ during LDL oxidation leads to an increased uptake of oxLDL by macrophages, which could be primarily attributed to the increased uptake of the oxidized phospholipids enriched lipid moiety of oxLDL.     

Guanine nucleotides reduce the free calcium requirement for secretion of granule constituents from permeabilized human neutrophils

Human neutrophils can be permeabilized with the cholesterol complexing agent digitonin and then induced to secrete lysosomal constituents by increases in free Ca²⁺ alone. In order of increasing requirements for Ca²⁺, vitamin B-12 binding protein, lysozyme and β-glucuronidase were released. A variety of guanine nucleotides were examined with respect to their abilities to modulate this response. GTP, along with its analogues 5′-guanylylimidodiphosphate (Gpp[NH]p) and guanosine-5′-O-[3-thio]-triphosphate (GTP[γS]) decreased the Ca²⁺ requirements for secretion of all three granule constituents by one third to one order of magnitude. This synergy was dependent upon the concentration of guanine nucleotides employed. The effects of Gpp[NH]p could be blocked with the inactive derivative GDP[β-S]. The active guanine nucleotides, particularly GTP, served as stimuli in their own right. At high concentrations of Ca²⁺ and GTP, degranulation was strikingly inhibited; inhibition was also achieved with high concentrations of guanylyl[β,γ-methylene]diphosphate (Gpp[CH₂]p). Both GDP and GMP were without any effect. When neutrophils were pretreated with pertussis toxin, granule discharge induced by fMet-Leu-Phe was almost completely blocked, as reported by others. If the neutrophils pretreated with pertussis toxin were then permeabilized with digitonin, the synergy between Ca²⁺ and the stimulatory guanine nucleotides was maintained. These data suggest the involvement of G-proteins in secretion induced by Ca²⁺; however, this response either uses a different G-protein or a different pool of G-proteins from those responses triggered by fMet-Leu-Phe.     

The transfer of VLDL-associated phospholipids to activated platelets depends upon cytosolic phospholipase A2 activity

We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 microM PL) to platelets (2 x 10(8)/ml) was measured after incubations of 1 h at 37 degrees C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 microM); esculetin, a 12-lipoxygenase inhibitor (20 microM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A(2) (PLA(2)) inhibitor (100 microM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca(2+) chelator (20 microM); bromoenol lactone (BEL), a Ca(2+)- independent phospholipase A(2) (iPLA(2)) inhibitor (100 nM); and 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 microM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A(2) (cPLA(2)) and iPLA(2), only cPLA(2) is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA(2)-dependent process. The inhibition of iPLA(2) by BEL had no effect on PL transfers.     

Inhibition of phospholipase A2 activity of guinea-pig alveolar macrophages by lipocortin-like proteins purified from mice lung

Guinea-pig alveolar macrophages were harvested by bronchoalveolar lavage and purified by differential adhesion. They were labeled with 14C-Arachidonic acid and then exposed to platelet-activating factor or to the calcium ionophore A23187. The activity of cellular phospholipase A2 was considered as the release of free 14C-Arachidonic acid in the cell supernatant. The pretreatment of guinea-pig alveolar macrophages with two lipocortin-like proteins (36 kDa and 40 kDa) purified from mice lung induced a significant inhibition of their phospholipase A2 activity upon platelet-activating factor and calcium ionophore stimulation. These results indicate that lipocortin-like proteins can modulate the phospholipase A2 activity of isolated cells in vitro.     

Recognition of acidic phospholipase A2 activity in plasma membranes of resident peritoneal macrophages

Phospholipase (PLase) activities in the plasma membrane of guinea pig peritoneal macrophages were studied, as these enzymes having such activity may be candidates for the release of arachidonic acid (AA) from phosphatidylcholine (PC). An AA release system operating at acidic pH was identified in the macrophage plasma membrane and characterized. This membrane-bound acidic PLase A2 had an optimum pH at 4.5, and enzyme activation was observed in Ca++-free medium; but the maximum activity was found at 0.5 mM Ca++ concentration. The Km value for PC of acidic PLase A2 was 4.2 microM, and a Michaelis-Menten relationship was evident. Calcium might act as a cofactor at some intermediate step during the activation of acidic PLase A2 in light of the uncompetitive manner of Ca++ action. Furthermore, the release of [3H]-AA from preradiolabelled macrophage plasma membranes occurred with the addition of Ca++ at pH 4.5. These data suggest that the acid PLase A2 is a component of the plasma membrane and is not due to lysosomal contamination since membrane-bound acidic PLase A2 properties are opposite to those found for lysosomal PLase A2.