Cytosolic phospholipase A2 and lysophospholipid acyltransferases

Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of ester bonds at sn-2 positions of glycerophospholipids (PL), producing free fatty acids and lysophospholipids. In mammals, the PLA₂ superfamily comprises more than 30 known enzymes, including various structurally and biochemically different enzymes with diverse biological functions. Some of the enzymes are involved in the production of lipid mediators, including eicosanoids and lysophospholipid-related lipid mediators. Among them, cytosolic PLA₂α (cPLA₂α), a member of cPLA₂ family, is one of the most important intracellular PLA₂s. Upon cell activation, cPLA₂α is activated and involved in eicosanoid production under various physiological and pathological conditions. PLA₂s also play a role in membrane PL remodeling by coupling with re-acylation processes mediated by lysophospholipid acyltransferases (LPLATs) to generate sn-1/sn-2 fatty acid asymmetry of PLs. This review summarizes the biochemical and in vivo roles of cPLA₂ enzymes and LPLATs, including results from animal and human studies.This article is part of a Special Issue entitled Novel functions of phospholipase A₂ Guest Editors: Makoto Murakami and Gerard Lambeau.     

Computer-aided drug design guided by hydrogen/deuterium exchange mass spectrometry: A powerful combination for the development of potent and selective inhibitors of Group VIA calcium-independent phospholipase A2

Potent and selective inhibitors for phospholipases A₂ (PLA₂) are useful for studying their intracellular functions. PLA₂ enzymes liberate arachidonic acid from phospholipids activating eicosanoid pathways that involve cyclooxygenase (COX) and lipoxygenase (LOX) leading to inflammation. Anti-inflammatory drugs target COX and LOX; thus, PLA₂ can also be targeted to diminish inflammation at an earlier stage in the process. This paper describes the employment of enzymatic assays, hydrogen/deuterium exchange mass spectrometry (DXMS) and computational chemistry to develop PLA₂ inhibitors. Beta-thioether trifluoromethylketones (TFKs) were screened against human GVIA calcium-independent, GIVA cytosolic and GV secreted PLA₂s. These compounds exhibited inhibition toward Group VIA calcium-independent PLA₂ (GVIA iPLA₂), with the most potent and selective inhibitor 3 (OTFP) obtaining an X_I(50) of 0.0002 mole fraction (IC₅₀ of 110 nM). DXMS binding experiments in the presence of OTFP revealed the peptide regions of GVIA iPLA₂ that interact with the inhibitor. Molecular docking and dynamics simulations in the presence of a membrane were guided by the DXMS data in order to identify the binding mode of OTFP. Clustering analysis showed the binding mode of OTFP that occupied 70% of the binding modes occurring during the simulation. The resulted 3D complex was used for docking studies and a structure–activity relationship (SAR) was established. This paper describes a novel multidisciplinary approach in which a 3D complex of GVIA iPLA₂ with an inhibitor is reported and validated by experimental data. The SAR showed that the sulfur atom is vital for the potency of beta-thioether analogues, while the hydrophobic chain is important for selectivity. This work constitutes the foundation for further design, synthesis and inhibition studies in order to develop new beta-thioether analogues that are potent and selective for GVIA iPLA₂ exclusively.     

Dexamethasone,a Specific PLA2 Inhibitor,Allows Nonpathogenic Pseudomonas fluorescens to Induce Virulence Against Spodoptera exigua

Phospholipase A₂ (PLA₂) catalyzes phospholipids at sn-2 position to release arachidonic acid (20:4n-6). The arachidonic acid is further oxidized to form different eicosanoids, which play biological mediators to express cellular or humoral immune reactions in response to pathogen infection. Xeno-rahbdus and Photorhabdus, the symbiotic bacteria of entomopathogenic nematodes, have been known to inhibit PLA₂ to express their pathogenicity. This research aimed to test a hypothesis that other entomopathogenic bacteria also inhibit PLA₂ to express their pathogenicity in Spodopera exigua. Two bacterial species of Enterococcus faecalis and Pseudomonas fluorescens presumably different in ento-mopathogenicity were analyzed in their PLA₂ inhibitory activities. A pathogenic E. faecalis induced significantly immunodepression of S. exigua by inhibiting PLA₂ activity because the bacteria-infected S. exigua recovered immune reactions after the addition of arachidonic acid. However, the nonpathogenic P. fluorescens did not induce immunodepression because the addition of arachidonic acid to P. fluorescens-infected S. exigua did not further increase immune capacities while dexamethasone, a PLA₂ inhibitor, could decrease the immune activities. Injection of E. faecalis along with 10 μg of dexamethasone significantly increased pathogenicity in comparison with the bacteria alone. Moreover, the addition of dexamethasone transformed nonpathogenic P. fluorescens into pathogenic bacterium. This study suggests an evidence that PLA₂ is an inhibitory target even for entomopathogenic bacteria not related with entomopathogenic nematodes, and that the inhibition of PLA₂ determines the bacterial virulence in S. exigua.     

Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms

Summary The amino acid sequences of 40 secreted phospholipase A₂'s (PLA₂) were aligned and a phylogenetic tree derived that has three main branches corresponding to elapid (group I), viperid (group II), and insect venom types of PLA₂. The human pancreatic and recently determined nonpancreatic sequences in the comparison align with the elapid and viperid categories, repectively, indicating that at least two PLA₂ genes existed in the vertebrate line before the divergence of reptiles and mammals about 200–300 million years ago. This allows resolution for the first time of major genetic events in the evolution of current PLA₂'s and the relationship of human PLA₂'s to those of snake venom, many of which are potent toxins. Implications for possible mechanisms of regulation of mammalian intra- and extracellular PLA₂'s are discussed, as well as issues relating to the search for the controlling enzymes in arachidonic acid release, prostaglandin generation, and signal transduction.     

Recent progress in phospholipase A₂ research: from cells to animals to humans

Mammalian genomes encode genes for more than 30 phospholipase A₂s (PLA₂s) or related enzymes, which are subdivided into several classes including low-molecular-weight secreted PLA₂s (sPLA₂s), Ca²⁺-dependent cytosolic PLA₂s (cPLA₂s), Ca²⁺-independent PLA₂s (iPLA₂s), platelet-activating factor acetylhydrolases (PAF-AHs), lysosomal PLA₂s, and a recently identified adipose-specific PLA. Of these, the intracellular cPLA₂ and iPLA₂ families and the extracellular sPLA₂ family are recognized as the “big three”. From a general viewpoint, cPLA₂α (the prototypic cPLA₂) plays a major role in the initiation of arachidonic acid metabolism, the iPLA₂ family contributes to membrane homeostasis and energy metabolism, and the sPLA₂ family affects various biological events by modulating the extracellular phospholipid milieus. The cPLA₂ family evolved along with eicosanoid receptors when vertebrates first appeared, whereas the diverse branching of the iPLA₂ and sPLA₂ families during earlier eukaryote development suggests that they play fundamental roles in life-related processes. During the past decade, data concerning the unexplored roles of various PLA₂ enzymes in pathophysiology have emerged on the basis of studies using knockout and transgenic mice, the use of specific inhibitors, and information obtained from analysis of human diseases caused by mutations in PLA₂ genes. This review focuses on current understanding of the emerging biological functions of PLA₂s and related enzymes.     

Effects of exogenous phospholipases on brain membrane phospholipid perturbation, (Na+ + K+)-ATPase activity and cellular swelling of brain slices

Rat brain membranes were incubated with bee venom phospholipase A₂ (PLA₂) or phospholipase C (PLC) from Clostridium perfringens. PLA₂ caused a significant increase in free polyunsaturated fatty acids concomitant with membrane phospholipid degradation as monitored by HPLC and by gas chromatography. Equal concentrations of PLC had a much lesser effect than PLA₂. Divergent and differential effects were shown on deacylation and incorporation of [³H]arachidonic acid in membrane phospholipids. The incorporation of [³H]arachidonic acid into various phospholipids was greatly reduced by PLA₂ (0.018 units/ml) whereas PLC at identical concentration was not effective. PLA₂ inhibited (Na⁺ + K⁺)-ATPase but was not effective on p-nitrophenyl-phosphatase activity whereas PLC stimulated both enzymes. PLA₂ induced swelling of cortical brain slices whereas PLC was not effective. Thus, the severity of the perturbation of membrane integrity, and the inhibition of (Na⁺ + K⁺)-ATPase in brain membranes may play an important role in cellular swelling of brain slices induced by PLA₂.     

Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta

We report on phospholipase A₂ (PLA₂) activity in homogenates prepared from fat bodies of the tobacco hornworm Manduca sexta. PLA₂ activity is responsible for hydrolyzing fatty acids from the sn-2 position of phospholipids. The rate of hydrolysis increased with increasing homogenate protein concentration up to ～? 320 μg protein/ml reaction volume. Higher protein concentrations did not appreciably increase the rate of PLA₂ activity. As seen in some, but not all PLA₂s from mammalian sources, hydrolyzing activity increased linearly with time. The fat body activity was sensitive to pH (optimal activity at pH 8–9) and temperature (optimal activity at ～?40°C). The activity was associated with fat body rather than hemolymph, because no activity was detected in cell-free serum. The fat body PLA₂ activity differs from the majority of PLA₂s with respect to calcium requirements. Whereas most PLA₂s are calcium-independent. A few others are known to require submicromolar calcium concentrations. The fat body activity appears to be calcium independent. These data show that a PLA₂ activity that can hydrolyze arachidonic acid from the sn-2 position of phospholipids is associated with the tobacco hornworm fat body. The biological significance of this activity relates to biosynthesis of eicosanoids. Pharmacological inhibition of PLA₂ impairs the ability of this insect to respond to bacterial infections. Since the impairment can be reversed by treatment with exogenous arachidonic acid, the PLA₂ activity may be an important step in eicosanoid biosynthesis. © 1993 Wiley-Liss, Inc.     

Emerging roles for phospholipase A2 enzymes in cancer

Phospholipase A₂ (PLA₂) enzymes (EC3.1.4.4) regulate the release of biologically active fatty acids and lysophospholipids from membrane phospholipid pools. These lipids are also substrates for intracellular biochemical pathways that generate potent autocrine and paracrine lipid mediators such as the eicosanoids and platelet activating factor. These factors, in turn, regulate cell proliferation, survival, differentiation, motility, tissue vascularisation, and immune surveillance in virtually all tissues, functions that are subverted by cancer cells for tumour growth and metastasis. Thus the relevance of PLA₂-dependent pathways to the genesis and progression of cancer has been of interest since their discovery and with recent technological advances, their role in tumourigenesis has become more tractable experimentally. Limited human genetic studies have not yet identified PLA₂ enzymes as classical mutated oncogenes or tumour suppressor genes. However, there is strong evidence that of the 22 identified human PLA₂ enzymes, ten of which have been studied in cancer to date, most are aberrantly expressed in a proportion of tumours derived from diverse organs. Correlative and functional studies implicate the expression of some secreted enzymes (sPLA₂s), particularly the best studied enzyme Group IIA sPLA₂ in either tumour promotion or inhibition, depending on the organ involved and the biochemical microenvironment of tumours. As in immune-mediated inflammatory pathologies, genetic deletion studies in mice, supported by limited studies with human cells and tissues, have identified an important role for Group IVA PLA₂ in regulating certain cancers. Pharmacological intervention studies in prostate cancer suggest that hGIIA-dependent tumour growth is dependent on indirect regulation of Group IVA PLA₂. Group VI calcium-independent PLA₂ enzymes have also been recently implicated in tumourigenesis with in vitro studies suggesting multiple possible roles for these enzymes. Though apparently complex, further characterization of the regulatory relationships amongst PLA₂ enzymes, lipid mediator biosynthetic enzymes and the lipid mediators they produce during tumour progression is required to define the biochemical context in which the enzymes modulate cancer growth and development.     

Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A2

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A₂ (PLA₂) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA₂s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA₂ activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA₂, cPLA₂. However, unlike the cPLA₂ enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA₂ activity is sensitive to inhibitors that block the activities of cPLA₂-α and the 80 kDa calcium-independent PLA₂, iPLA₂, that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [³H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA₂ that may play a role in the basal phospholipid metabolism of these cells.     

Isozymes inhibited by active site blocking: versatility of calcium indifferent hesperidin binding to phospholipase A2 and its significance

sPLA₂ is released under inflammatory conditions from neutrophils, basophils and T-cells. They cleave the cellular phospholipids leading to the release of arachidonic acid and there by provide intermediates for biosynthesis of inflammatory mediators. The focus of this study is on the interaction of hesperidin, a natural flavonoid with Group IB, IIA, and V and X isozymes of sPLA₂. Affinity of hesperidin towards PLA₂ isozymes was analyzed through enzymatic studies and molecular modeling. The experiments showed that hesperidin competitively inhibited PLA₂ with IC₅₀ of 5.1?µM. Molecular modeling studies revealed the association of hesperidin with the docking scores ?6.90, ?9.53, ?5.63 and ?8.29?kcal for isozymes Group IB, IIA, V and X of PLA₂ respectively. Their binding energy values were calculated as ?20.25, ?21.63, ?21.66 and ?33.43?kcal for the Group IB, IIA, V and X respectively. Structural model for Group V was made by homology modeling since no structural coordinates were available. Molecular dynamics studies were carried out to evaluate the structural stability of protein ligand complex. The analyses showed that hesperidin blocked the entry of the substrate to the active site of PLA₂ and it was indifferent to the differences of the isozymes. Hence, hesperidin might serve as lead for designing highly specific anti-inflammatory drugs directed to the PLA₂ isozyme specific to various diseases, with IC₅₀ value of therapeutic significance.     

Leukotriene and prostaglandin production in rat brain synaptosomes treated with phospholipase A2 neurotoxins and enzymes

β-Bungarotoxin (β-BuTX) and notexin cause an irreversible blockade of neurotransmitter release through specific and potent effects at the presynaptic nerve terminal, however, the mechanism of action in uncertain. We examined the effects of β-BuTX and notexin on LT and PG production in rat cerebrocortical synaptosomes in order to determine if eicosanoid production might mediate or regulate the pharmacological actions of these phospholipase A₂(PLA₂) neurotoxins. The effects of the PLA₂ enzymes isolated from Naja naja atra and Naja nigricollis snake venoms (which are not presynaptic selective) on LT and PG production were compared with the effects of β-BuTX and notexin. N. n. atra PLA₂, β-BuTX, and notexin (all 50 nM) produced a time dependent rise in free fatty acids as measured in synaptic plasma membranes isolated from treated synaptosomes. Both the PLA₂ neurotoxins and enzymes stimulated LTC₄, LTB₄, and PGE₂ production, as measured by radioimmunoassay. In all cases, the PLA₂ enzymes were more potent than the PLA₂ neurotoxins. This observations correlates with their relative enzymatic potencies, as measured by free fatty acid generation. EDTA and BSA antagonized PLA₂ induced LTB₄ production and BSA also antagonized PLA₂ induced PGE₂ production. These results suggest that stimulation of eicosanoid production does not mediate the potent and specific presynaptic actions of β-BuTX and notexin.     

Structural and phylogenetic basis for the classification of group III phospholipase A2

Secretory phospholipase A₂ (PLA₂) catalyses the hydrolysis of the sn-2 position of glycerophospholipids to liberate arachidonic acid, a precursor of eicosanoids, that are known mediators of inflammation. The group III PLA₂ enzymes are present in a wide array of organisms across many species with completely different functions. A detailed understanding of the structure and evolutionary proximity amongst the enzymes was carried out for a meaningful classification of this group. Fifty protein sequences from different species of the group were considered for a detailed sequence, structural and phylogenetic studies. In addition to the conservation of calcium binding motif and the catalytic histidine, the sequences exhibit specific ‘amino acid signatures’. Structural analysis reveals that these enzymes have a conserved globular structure with species specific variations seen at the active site, calcium binding loop, hydrophobic channel, the C-terminal domain and the quaternary conformational state. Character and distance based phylogenetic analysis of these sequences are in accordance with the structural features. The outcomes of the structural and phylogenetic analysis lays a convincing platform for the classification the group III PLA₂s into (1A) venomous insects; (IB) non-venomous insects; (II) mammals; (IIIA) gila monsters; (IIIB) reptiles, amphibians, fishes, sea anemones and liver fluke, and (IV) scorpions. This classification also helps to understand structure-function relationship, enzyme-substrate specificity and designing of potent inhibitors against the drug target isoforms.     

Role of Cytosolic Phospholipase A2 in Oxidative and Inflammatory Signaling Pathways in Different Cell Types in the Central Nervous System

Phospholipases A₂ (PLA₂s) are important enzymes for the metabolism of fatty acids in membrane phospholipids. Among the three major classes of PLA₂s in the mammalian system, the group IV calcium-dependent cytosolic PLA₂ alpha (cPLA₂α) has received the most attention because it is widely expressed in nearly all mammalian cells and its active participation in cell metabolism. Besides Ca²⁺ binding to its C2 domain, this enzyme can undergo a number of cell-specific post-translational modifications, including phosphorylation by protein kinases, S-nitrosylation through interaction with nitric oxide (NO), as well as interaction with other proteins and lipid molecules. Hydrolysis of phospholipids by cPLA₂ yields two important lipid mediators, arachidonic acid (AA) and lysophospholipids. While AA is known to serve as a substrate for cyclooxygenases and lipoxygenases, which are enzymes for the synthesis of eicosanoids and leukotrienes, lysophospholipids are known to possess detergent-like properties capable of altering microdomains of cell membranes. An important feature of cPLA₂ is its link to cell surface receptors that stimulate signaling pathways associated with activation of protein kinases and production of reactive oxygen species (ROS). In the central nervous system (CNS), cPLA₂ activation has been implicated in neuronal excitation, synaptic secretion, apoptosis, cell-cell interaction, cognitive and behavioral function, oxidative-nitrosative stress, and inflammatory responses that underline the pathogenesis of a number of neurodegenerative diseases. However, the types of extracellular agonists that target intracellular signaling pathways leading to cPLA₂ activation among different cell types and under different physiological and pathological conditions have not been investigated in detail. In this review, special emphasis is given to metabolic events linking cPLA₂ to activation in neurons, astrocytes, microglial cells, and cerebrovascular cells. Understanding the molecular mechanism(s) for regulation of this enzyme is deemed important in the development of new therapeutic targets for the treatment and prevention of neurodegenerative diseases.     

Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge

We propose that expression of four genes encoding secretory phospholipases A₂ (sPLA₂) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA₂-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA₂ activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA₂ and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA₂s. The recombinant sPLA₂s were inhibited by sPLA₂ inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA₂ genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA₂s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA₂s showed the presence of the sPLA₂ enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA₂ genes that mediate nodulation reactions strongly supports our hypothesis that sPLA₂s are central enzymes in insect cellular immune reactions.     

Analogs of arachidonic acid methylated at C-7 and C-10 inhibit leukotriene biosynthesis and phospholipase A2 activity

The ability of (all Z)-7,7-dimethyl-5,8,11,14-eico-satetraenoic acid, (all Z)-7,7-dimethyl-5,8,11-eicosatrienoic acid, (Z,Z)-7,7-dimethyl-5,8-eicosadienoic acid, (all Z)-10,10-dimethyl-5,8,11,14-eicosatetraenoic acid, (all Z)-10,10-dimethyl-5,8,11-eicosatrienoic acid, and rac-(Z,Z)-15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid to inhibit ionophore-induced slow-reacting substance of anaphylaxis (SRS-A) biosynthesis in rat peritoneal cells was studied. It was thought that compounds such as these might inhibit proton abstractions at the 7 or 10 carbon positions on arachidonic acid which are thought to be important in the mechanism of catalysis of Δ⁵-lipoxygenase(Δ⁵-LO). All compounds were found to be potent inhibitors of SRS-A biosynthesis in the in vitro rat peritoneal cell system (IC₅₀ < 10 μM). In fact they were more potent inhibitors in the test system than standard Δ⁵-LO inhibitors such as NDGA and quercetin. To determine if the mechanism of inhibition of the dimethyl arachidonic acid analogs did involve gD⁵-LO inhibition these compounds were evaluated in an assay system utilizing the Δ⁵-LO from rat basophilic leukemia (RBL^?1_cells. It was found, however, that these compounds were much less potent inhibitors of this enzyme (IC₅₀ ～ 100 μM) than standard compounds such as NDGA (IC₅₀ 0.14 μM) and quercetin (IC₅₀, 0.2 μM). The arachidonic acid analogs were subsequently found to be potent inhibitors of phospholipase A₂ (PLA₂) enzymes with IC₅₀'s between 10–20 μM as inhibitors of a snake venom enzyme. In fact these compounds are among the most potent inhibitors of PLA₂ yet studied, having potencies better than standards such as p-bromophenacyl bromide (IC₅₀, 87 μM) and U-10029A (IC₅₀, 36 μM). These results suggest that the methylated arachidonic acid analogs may inhibit SRS-A biosynthesis through inhibiting PLA₂.     

A chromogenic substrate for solid-phase detection of phospholipase A2

A method for solid-phase detection of phospholipase A₂ (PLA₂) was developed. The method uses 1-octanoyloxynaphthalene-3-sulfonic acid, which was found to be a good substrate of PLA₂. The substrate is hydrolyzed by PLA₂ into 1-naphthol-3-sulfonic acid, which is spontaneously coupled with coexisting diazonium salt to form a red-purple azo dye. Streptomyces and bovine pancreatic PLA₂ spotted on a nitrocellulose membrane could be detected by this method with considerable sensitivity. In addition, colonies of recombinant Escherichia coli producing bacterial PLA₂ were distinguishable from those producing an inactive mutant PLA₂, facilitating high-throughput screening in directed evolution of the enzyme.     

PLA2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators

Phospholipase A₂ (PLA₂) is an enzyme which participates in signalling mechanisms cleaving arachidonate from sn-2 position of glycerophospholipids. In this study we have verified the existence of a PLA₂-like activity in the free living protozoan, Tetrahymena pyriformis GL. This activity is Ca²⁺-independent, EDTA (10 mM) has no effect on its activity. Quinacrine (0.1 mM) and 4-bromophenacyl bromide (BPB; 0.1 mM) inhibited, melittin (20 μg/ml significantly stimulated the PLA₂ activity and the release of free arachidonic acid (AA) from 1-acyl 2-¹⁴C-arachidonyl-3-phosphatidylethanolamine substrate. Melittin stimulated PLA₂ hyperactivity is Ca²⁺-dependent. There was no considerable alteration in the PLA₂ activity by stimulation of the activity by tyrosine kinase (with vanadate, H₂0₂), phospholipase C (PLC) (with phorbol 12, 13-dibutyrate) or G-proteins (with NaF, AlF₄ thus in Tetrahymena PLA₂ activity seems to be independent of these—in Tetrahymena (also functioning)—signalling pathways. Treatment with quinacrine and BPB leads to decreased synthesis and disturbed breakdown of phospholipids and phosphoinositides. These findings suggest that PLA₂ activity is in connection with the phospholipid metabolism of Tetrahymena.     

Phospholipase A2 in hemocytes of the tobacco hornworm,Manduca sexta

On the hypothesis that prostaglandins and other eicosanoids mediate nodulation responses to bacterial infections in insects, we describe an intracellular phospholipase A₂ (PLA₂) in homogenates prepared from hemocytes collected from the tobacco hornworm, Manduca sexta. PLA₂ hydrolyzes fatty acids from the sn-2 position of phospholipids. Some PLA₂s are thought to be the first and rate-limiting step in biosynthesis of prostaglandins and other eicosanoids. The hemocyte PLA₂ activity was sensitive to hemocyte homogenate protein concentration (up to 250 μg protein/reaction), pH (optimal activity at pH 8.0), and the presence of a Ca²⁺ chelator. Like PLA₂s from mammalian sources, the hemocyte PLA₂ was inhibited by the phospholipid analog oleyoxyethyl phosphorylcholine. Whereas most intracellular PLA₂s require Ca²⁺ for catalytic activity, some PLA₂s, including the hemocyte enzyme, are Ca²⁺-independent. The hemocyte PLA₂ exhibited a preference for arachidonyl-associated substrate over palmitoyl-associated substrate. These findings show that M. sexta hemocytes express a PLA₂ that shows a marked preference for hydrolyzing arachidonic acid from phospholipids. The biological significance of this enzyme relates to cellular immune responses to bacterial infections. The hemocyte PLA₂ may be the first biochemical step in synthesis of the eicosanoids that mediate cellular immunity in insects. © 1996 Wiley-Liss, Inc.     

Preliminary Characterization of Phospholipase A2 in Lagenidium giganteum

MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A₂ in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A₂ (PLA₂) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA₂ activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA₂ activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1-¹⁴C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ all enhanced PLA² activity, while Co²⁺, Fe²⁺, and Zn²⁺ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca²⁺ and Mn²⁺ than Mg²⁺, reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA₂ activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA₂ hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA₂ activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA₂ activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.     

Phospholipase A2 in astrocytes

Astrocytes comprise the major cell type in the central nervous system (CNS) and they are essential for support of neuronal functions by providing nutrients and regulating cell-to-cell communication. Astrocytes also are immune-like cells that become reactive in response to neuronal injury. Phospholipases A₂ (PLA ₂) are a family of ubiquitous enzymes that degrade membrane phospholipids and produce lipid mediators for regulating cellular functions. Three major classes of PLA ₂ are expressed in astrocytes: group IV calcium-dependent cytosolic PLA ₂ (cPLA₂), group VI calcium-independent PLA ₂ (iPLA₂), and group II secretory PLA ₂ (sPLA₂). Upregulation of PLA ₂ in reactive astrocytes has been shown to occur in a number of neurodegenerative diseases, including stroke and Alzheimer’s disease. This review focuses on describing the effects of oxidative stress, inflammation, and activation of G protein-coupled receptors on PLA ₂ activation, arachidonic acid (AA) release, and production of prostanoids in astrocytes.