Hydrolysis of minor glycerophospholipids of plasma lipoproteins by human group IIA, V and X secretory phospholipases A2

We investigated the hydrolysis of the minor glycerophospholipids of human HDL(3), total HDL and LDL using human group IIA, V and X secretory phospholipases A(2) (sPLA(2)s). For this purpose we employed the enzyme and substrate concentrations and incubation times optimized for hydrolysis of phosphatidylcholine (PtdCho), the major glycerophospholipid of plasma lipoproteins. In contrast to PtdCho, which was readily hydrolyzed by group V and X sPLA(2)s, and to a lesser extent by group IIA sPLA(2), the minor ethanolamine, inositol and serine glycerophospholipids exhibited marked resistance to hydrolysis by all three sPLA(2)s. Thus, when PtdCho was hydrolyzed about 80%, the ethanolamine and inositol glycerophospholipids reached a maximum of 40% hydrolysis. The hydrolysis of phosphatidylserine (PtdSer), which was examined to a more limited extent, showed similar resistance to group IIA, V and X sPLA(2)s, although the group V sPLA(2) attacked it more readily than group X sPLA(2) (52% versus 39% hydrolysis, respectively). Surprisingly, the group IIA sPLA(2) hydrolysis remained minimal at 10-15% for all minor glycerophospholipids, and was of the order seen for the PtdCho hydrolysis by group IIA sPLA(2) at the 4-h digestion time. All three enzymes attacked the oligo- and polyenoic species in proportion to their mole percentage in the lipoproteins, although there were exceptions. There was evidence of a more rapid destruction of the palmitoyl compared to the stearoyl arachidonoyl glycerophospholipids. Overall, the characteristics of hydrolysis of the molecular species of the lipoprotein-bound diradyl GroPEtn, GroPIns and GroPSer by group V and X sPLA(2)s differed significantly from those observed with lipoprotein-bound PtdCho. As a result, the acidic inositol and serine glycerophospholipids accumulated in the digestion residues of both LDL and HDL, and presumably increased the acidity of the residual particles. An accumulation of the ethanolamine glycerophospholipids in the sPLA(2) digestion residues also had not been previously reported. These results further emphasize the diversity in the enzymatic activity of the group IIA, V and X sPLA(2)s. Since these sPLA(2)s possess comparable tissue distribution, their combined activity may exacerbate their known proinflammatory and proatherosclerotic function.     

Role of sphingomyelin and ceramide in the regulation of the activity and fatty acid specificity of group V secretory phospholipase A2

We previously showed that group V secretory phospholipase A(2) (sPLA(2)V) is inhibited by sphingomyelin (SM), but activated by ceramide. Here, we investigated the effect of sphingolipid structure on the activity and acyl specificity of sPLA(2)V. Degradation of HDL SM to ceramide, but not to ceramide phosphate, stimulated the activity by 6-fold, with the release of all unsaturated fatty acids being affected equally. Ceramide-enrichment of HDL similarly stimulated the release of unsaturated fatty acids. Incorporation of SM into phosphatidylcholine (PC) liposomes preferentially inhibited the hydrolysis of 16:0-20:4 PC. Conversely, SMase C treatment or ceramide incorporation resulted in preferential stimulation of hydrolysis of 16:0-20:4 PC. The presence of a long chain acyl group in ceramide was essential for the activation, and long chain diacylglycerols were also effective. However, ceramide phosphate was inhibitory. These studies show that SM and ceramide in the membranes and lipoproteins not only regulate the activity of phospholipases, but also the release of arachidonate, the precursor of eicosanoids.     

Regulation of the expression of group IIA and group V secretory phospholipases A(2) in rat mesangial cells

Rat mesangial cells synthesize and secrete a secretory phospholipase A(2) upon stimulation of the cells with cytokines, like IL-1beta and TNF and with cAMP elevating agents like forskolin. This enzyme was previously characterized to belong to group IIA sPLA(2). The discovery of several other low molecular weight phospholipases, like group IIC in murine testis and group V in human and rat heart, prompted investigations on the presence of group IIC and group V sPLA(2) in rat mesangial cells. This was done by isolating the RNA from stimulated cells and performing RT-PCR, using primers specific for group IIC and V sPLA(2). The results indicate that rat mesangial cells upon stimulation express next to group IIA also group V sPLA(2). No indications were obtained for the expression of group IIC sPLA(2). The regulation of the expression of group V sPLA(2) at the mRNA level was further investigated by examining the time-dependent expression, the influence of dexamethasone and the signaling route of the IL-1beta stimulation. The results show that the IL-1beta induced expression of group V sPLA(2) mRNA was time dependent and, similar to that of group IIA sPLA(2) mRNA, involves activation of NF-kappaB. However, in contrast to the group IIA sPLA(2), the expression of group V sPLA(2) was not influenced by the presence of dexamethasone. The expression of both phospholipases was also examined at the protein level in stimulated mesangial cells. Western blot analysis shows that stimulated mesangial cells synthesize both group IIA and group V sPLA(2) protein but the expression of group V is lower compared to that of group IIA sPLA(2). In addition, the extent of secretion into the medium appears to be considerably higher for group IIA than for group V sPLA(2).     

Systematic evaluation of transcellular activities of secretory phospholipases A2. High activity of group V phospholipases A2 to induce eicosanoid biosynthesis in neighboring inflammatory cells

The mechanisms by which secretory phospholipase A2 (PLA2) exerts cellular effects are not fully understood. To elucidate these mechanisms, we systematically and quantitatively assessed the activities of human group IIA, V, and X PLA2s on originating and neighboring cells using orthogonal fluorogenic substrates in various mixed cell systems. When HEK293 cells stably expressing each of these PLA2s were mixed with non-transfected HEK293 cells, group V and X PLA2s showed strong transcellular lipolytic activity, whereas group IIA PLA2 exhibited much lower transcellular activity. The transcellular activity of group V PLA2 was highly dependent on the presence of cell surface heparan sulfate proteoglycans of acceptor cells. Activation of RBL-2H3 and DLD-1 cells that express endogenous group V PLA2 led to the secretion of group V PLA2 and its transcellular action on neighboring human neutrophils and eosinophils, respectively. Similarly, activation of human bronchial epithelial cells, BEAS-2B, caused large increases in arachidonic acid and leukotriene C4 release from neighboring human eosinophils. Collectively, these studies show that group V and X PLA2s can act transcellularly on mammalian cells and suggest that group V PLA2 released from neighboring cells may function in triggering the activation of inflammatory cells under physiological conditions.     

Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model.

Studies of the specificity of phospholipases A2 (PLA2s) for different substrates have usually been carried out in vesicles or mixed micelles, where differences in shape, size, or charge of vesicles formed with different phospholipids may give misleading results. Another factor is binding of the enzyme to the phospholipid surface, which has recently been addressed using vesicles of an anionic phospholipid, dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) to which some extracellular PLA2s were shown to bind with a very high affinity (Jain, M. K., and Berg, O. G. (1989) Biochem. Biophys. Acta 1002, 127-156). In the present report we have used a similar system to study the substrate preferences of two human PLA2s that are thought to be physiologically relevant in the metabolism of arachidonic acid: a recombinant form of the human synovial fluid (14 kDa) PLA2 and the cytosolic (85 kDa) PLA2 found in monocytic cells. It is shown that both human enzymes bind tightly to DMPM vesicles and follow the basic characteristics of processive hydrolysis in this model using analysis of progress curves and substrate competition experiments. Mixed vesicles containing DMPM with small amounts (3-5 mol%) of other phospholipids have been used to study the substrate selectivity of the two human isoenzymes. The synovial fluid PLA2 shows a clear preference (approximately 7-fold) for sn-glycero-3-phosphoethanolamine over sn-glycero-3-phosphocholine. Within glycerophosphocholines, this enzyme displays little preference for the sn-2 fatty acyl group, and a slight preference for phospholipids with sn-1-acyl versus sn-1-alkyl substituents. In contrast, the cytosolic PLA2 shows a marked selectivity for arachidonoyl in the sn-2 position and only minor differences in selectivity for the polar head group in the sn-3 position. This enzyme does not distinguish between sn-1-acyl and sn-1-alkyl subclasses of glycerophosphocholines.     

Differences between group X and group V secretory phospholipase A2 in lipolytic modification of lipoproteins

Secretory phospholipases A(2) (sPLA(2)s) are a diverse family of low molecular mass enzymes (13-18 kDa) that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA(2) (sPLA(2)-X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA(2) (sPLA(2)-V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA(2)-X in several respects. Although sPLA(2)-V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA(2)-X. In addition, the requirement of Ca(2+) for the lipolysis of LDL was about 10-fold higher for sPLA(2)-V than sPLA(2)-X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA(2)-V in the presence of sodium citrate, which contrasted with the potent response to sPLA(2)-X. Moreover, sPLA(2)-X, but not sPLA(2)-V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA(2)-X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (K(d) = 3.1 nM) in the presence of Ca(2+). Selective interaction of sPLA(2)-X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.     

Bactericidal properties of group IIA and group V phospholipases A2

Group V phospholipase A(2) (PLA(2)) is a recently characterized 14-kDa secretory PLA(2) of mammalian heart and macrophage-derived cells. Group IIA PLA(2), which is structurally close to group V PLA(2), has been shown to kill Gram-positive bacteria in vitro and to prevent symptoms of Gram-positive infection in vivo. We studied the antibacterial properties of fully active recombinant rat group IIA and V PLA(2)s. Both group IIA and V PLA(2)s were highly bactericidal against Gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Only high concentrations of group IIA PLA(2) showed some bactericidal effect against the Gram-negative bacterium Escherichia coli. Our results confirm that group IIA PLA(2) is a potent antibacterial enzyme against Gram-positive bacteria. Moreover, we show here that group V PLA(2) is a novel antibacterial mammalian protein, but is less potent than group IIA PLA(2). Both enzymes may be considered as future therapeutic agents against bacterial infections.     

Role of thyroid hormones in regulation of sphingomyelin metabolism in the liver.

Inhibition of thyroid gland function in rats with mercasolil sharply decreased thyroxine and triiodothyronine levels in blood serum and increased acid sphingomyelinase activity and sphingomyelin content in liver. Thyroxine injected into hypothyroid rats normalized the sphingomyelin content, reduced the free ceramide content, and further increased the acid sphingomyelinase activity in liver. Thyroxine stimulated de novo sphingomyelin synthesis. Changing the thyroid status of the rats did not influence the free sphingosine content. Thyroxine blocks the accumulation of free sphingosine in the liver during activation of sphingomyelinases.     

Role of membrane oxidation in controlling the activity of human group IIa secretory phospholipase A2 toward apoptotic lymphoma cells

The membranes of healthy lymphocytes normally resist hydrolysis by secretory phospholipase A₂. However, they become susceptible during the process of apoptosis. Previous experiments have demonstrated the importance of certain physical changes to the membrane during cell death such as a reduction in membrane lipid order and exposure of phosphatidylserine on the membrane surface. Nevertheless, those investigations also showed that at least one additional factor was required for rapid hydrolysis by the human group IIa phospholipase isozyme. This study was designed to test the possibility that oxidation of membrane lipids is the additional factor. Flow cytometry and confocal microscopy with a fluorescent probe of oxidative potential suggested that oxidation of the plasma membrane occurs during apoptosis stimulated by thapsigargin. When oxidative potential was high, the activity of human group IIa secretory phospholipase A₂ was enhanced 30- to 100-fold compared to that observed with conditions sufficient for maximal hydrolysis by other secretory phospholipase A₂ isoforms. Direct oxidation of cell membranes with either of two oxidizing agents also stimulated hydrolysis by secretory phospholipase A₂. Both oxidizers caused externalization of phosphatidylserine, but a change in lipid order did not always occur. These results demonstrated that membrane oxidation strongly stimulates human group IIa secretory phospholipase A₂ activity toward apoptotic cells. Interestingly, the change in membrane order, previously thought to be imperative for high rates of hydrolysis, was not required when membrane lipids were oxidized. Whether phosphatidylserine exposure is still necessary with oxidation remains unresolved since the two events could not be deconvoluted.     

Role of calcium-independent phospholipases (iPLA(2)) in phosphatidylcholine metabolism

The proposed role of calcium-independent phospholipase A(2) (iPLA(2)) in membrane phospholipid homeostasis was tested by examining the perturbation of phosphatidylcholine metabolism by enzyme overexpression. There are alternatively spliced forms of murine iPLA(2) that were widely expressed in mouse tissues: a long form containing exon-9 that is membrane-associated and a short form lacking exon-9 that is distributed between the membrane and cytosolic fractions. Enforced expression of either iPLA(2) isoform led to a significant increase in intracellular free fatty acid, lysophosphatidylcholine, and GPC without a concomitant increase in the incorporation of either exogenous arachidonic acid or choline. The accumulation of lysophosphatidylcholine in iPLA(2)-expressing cells illustrates the limited capacity of cells for reacylation and degradation of lysophospholipids. Since iPLA(2) overexpression did not accelerate either phospholipid remodeling or phosphatidylcholine synthesis, this enzyme does play a determinant (rate-controlling?) role in either of these cellular processes.     

Role of secretory and cytosolic phospholipase A(2) enzymes in lysophosphatidylcholine-stimulated monocyte arachidonic acid release

To determine if lysophosphatidylcholine (lysoPC) is able to induce proinflammatory changes in monocytes, its ability to stimulate arachidonic acid (AA) release, a product of phospholipase A2 (PLA(2)) activity, has been analyzed. LysoPC increased AA release in THP-1 and Mono Mac6 cells in a time- and concentration-dependent manner. The monocytes expressed both secretory and cytosolic PLA(2) enzymes and AA release was strongly reduced by cellular pretreatment with different PLA(2) inhibitors and by pertussis toxin, an inhibitor of G(i)-protein activation. This indicates that both cytosolic and secretory PLA(2) enzymes regulate specific lysoPC receptor-induced AA release, suggesting lysoPC participation in monocyte proinflammatory activation.     

Structure, function, and regulation of group V phospholipase A(2)

The hydrolysis of membrane phospholipid by phospholipase A(2) (PLA(2)) is a key step in the production of inflammatory eicosanoids. Recent cell studies have shown that secretory group V PLA(2) (gVPLA(2)) is involved in agonist-induced eicosanoid biosynthesis in mouse P388D1 cell line, mast cells, and transfected HEK 293 cells. gVPLA(2) is homologous to other group II PLA(2) family members but has distinctive enzymatic properties, including its activity to effectively hydrolyze phosphatidylcholine (PC) vesicles and the outer plasma membrane of mammalian cells. Mutational studies showed that gVPLA(2) has a unique structure that allows effective binding to PC membranes and efficient catalysis of an active-site-bound PC substrate. Thanks to this unique structure and activity, exogenously added gVPLA(2) can induce the eicosanoid biosynthesis in unstimulated inflammatory cells, including human neutrophils and eosinophils, suggesting that it might be able to trigger inflammatory responses under certain physiological conditions. Extensive structure-function and cell studies showed that gVPLA(2) could act directly on the outer plasma membranes of neutrophils and eosinophils. The release of fatty acids and lysophospholipids from the cell surfaces induces the translocation and activation of cytosolic PLA(2) and 5-lipoxygenase, resulting in the leukotriene synthesis. In case of neutrophils, induction of leukotriene B(4) synthesis by gVPLA(2) leads to the phosphorylation of cytosolic PLA(2) by a leukotriene B(4) receptor and MAP kinase-mediated mechanism. Finally, heparan sulfate proteoglycans in neutrophils appear to play a role of internalizing and degrading the cell surface-bound gVPLA(2) to protect the cells from extensive lipolytic damage.     

Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase

LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A₂ (sPLA₂-IIa and sPLA₂-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA₂-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA₂-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA₂-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis.     

Internalized group V secretory phospholipase A2 acts on the perinuclear membranes.

Mammalian secretory phospholipases A(2) (sPLA(2)) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA(2) mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA(2) acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA(2)s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA(2) hydrolysis products or cytokines, are necessary for the internalization of sPLA(2) into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA(2) internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA(2) induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA(2) acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis.     

Assay of phospholipases A(2) and their inhibitors by kinetic analysis in the scooting mode

Several cellular processes are regulated by interfacial catalysis on biomembrane surfaces. Phospholipases A(2) (PLA(2)) are interesting not only as prototypes for interfacial catalysis, but also because they mobilize precursors for the biosynthesis of eicosanoids and platelet activating factor, and these agents ultimately control a wide range of secretory and inflammatory processes. Since PLA(2) carry out their catalytic function at membrane surfaces, the kinetics of these enzymes depends on what the enzyme 'sees' at the interface, and thus the observed rate is profoundly influenced by the organization and dynamics of the lipidwater interface ('quality of the interface'). In this review we elaborate the advantages of monitoring interfacial catalysis in the scooting mode, that is, under the conditions where the enzyme remains bound to vesicles for several thousand catalytic turnover cycles. Such a highly processive catalytic turnover in the scooting mode is useful for a rigorous and quantitative characterization of the kinetics of interfacial catalysis. This analysis is now extended to provide insights into designing strategy for PLA(2) assays and screens for their inhibitors.     

Bacterial cell membrane hydrolysis by secreted phospholipases A(2): a major physiological role of human group IIa sPLA(2) involving both bacterial cell wall penetration and interfacial catalysis

The ability of human group IIa secreted phospholipase A(2) (human sPLA(2)) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA(2)s. This specificity is due to selective penetration of the highly cationic human sPLA(2)50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA(2) activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA(2) as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA(2)s. Overall, the results highlight the potential of the human sPLA(2) as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected.     

Redundant and segregated functions of granule-associated heparin-binding group II subfamily of secretory phospholipases A2 in the regulation of degranulation and prostaglandin D2 synthesis in mast cells

We herein demonstrate that mast cells express all known members of the group II subfamily of secretory phospholipase A2 (sPLA2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA2-IIA, -V, and -IID), but not heparin-nonbinding (sPLA2-IIC), enzymes released more granule-associated markers (beta-hexosaminidase and histamine) than mock- or cytosolic PLA2alpha (cPLA2alpha)-transfected cells after stimulation with IgE and Ag. Site-directed mutagenesis of sPLA2-IIA and -V revealed that both the catalytic and heparin-binding domains are essential for this function. Confocal laser and electron microscopic analyses revealed that sPLA2-IIA, which was stored in secretory granules in unstimulated cells, accumulated on the membranous sites where fusion between the plasma membrane and granule membranes occurred in activated cells. These results suggest that the heparin-binding sPLA2s bind to the perigranular membranes through their heparin-binding domain, and lysophospholipids produced in situ by their enzymatic action may facilitate the ongoing membrane fusion. In contrast to the redundant role of sPLA2-IIA, -IID, and -V in the regulation of degranulation, only sPLA2-V had the ability to markedly augment IgE/Ag-stimulated immediate PGD2 production, which reached a level comparable to that elicited by cPLA2alpha. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.     

Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2

Human groups IIA, V and X secretory phospholipases A2 (sPLA2s) were incubated with human HDL3, total HDL and LDL over a range of enzyme and substrate concentrations and exposure times. The residual phosphatidylcholines (PtdChos) were assayed by high performance liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). The enzymes varied markedly in their rates of hydrolysis of the different molecular species and in the production of lysoPtdCho. The sPLA2s were compared at a concentration of 1 microg/ml and an incubation time of 4 h, when all three enzymes showed significant activity. The groups V and X sPLA2 were up to 20 times more reactive than group IIA sPLA2. Group X sPLA2 hydrolyzed arachidonate and linoleate containing species preferentially, while group V hydrolyzed the linoleates in preference to polyunsaturates. In all instances, the arachidonoyl and linoleoyl palmitates were hydrolyzed in preference to the corresponding stearates by group X sPLA2. The group IIA enzyme appeared to hydrolyze randomly all diacyl molecular species. The minor alkylacyl and alkenylacyl glycerophosphocholines (GroPChos) were poor substrates for groups V and X sPLA2s and these phospholipids tended to accumulate. The present study demonstrates a preferential release of arachidonate from plasma lipoprotein PtdCho by group X sPLA2, as well as a relative resistance of polyunsaturated PtdChos to hydrolysis by group V enzyme, which had not been previously documented. The use of lipoprotein PtdCho as substrate with LC/ESI-MS identification of hydrolyzed molecular species eliminates much of the uncertainty about sPLA2 specificity arising from past analyses of fatty acid release from unknown or ill-defined sources.