Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

To better understand the principles underlying the substrate specificity of A-type phospholipases (PLAs), a high throughput mass spectrometric assay was employed to study the effect of acyl chain length and unsaturation of phospholipids on their rate of hydrolysis by three different secretory PLAs in micelles and vesicle bilayers. With micelles, each enzyme responded differently to substrate acyl chain unsaturation and double bond position, probably reflecting differences in the accommodative properties of their substrate binding sites. Experiments with saturated acyl positional isomers indicated that the length of the sn2 chain was more critical than that of the sn1 chain, suggesting tighter association of the former with the enzyme. Only the first 9–10 carbons of the sn2 acyl chain seem to interact intimately with the active site. Strikingly, no discrimination between positional isomers was observed with vesicles, and the rate of hydrolysis decreased far more with increasing chain length than with micelles, suggesting that translocation of the phospholipid substrate to the active site is rate-limiting with bilayers. Supporting this conclusion, acyl chain structure affected hydrolysis and spontaneous intervesicle transfer, which correlates with lipid efflux propensity, analogously. We conclude that substrate efflux propensity plays a more important role in the specificity of secretory PLA₂s than commonly thought and could also be a key attribute in phospholipid homeostasis in which (unknown) PLA₂s are key players.     

Modulation of eicosanoid biosynthesis and inhibition of substrate availability for phospholipase A2 by a modified hexose sugar, amiprilose hydrochloride

The modified hexose, sugar, amiprilose HCl [1, 2-O-isopropylinine-3-O-3′-(N′,N′-dimethylamino-n-propyl)-D-glucufuranose hydrochloride], has previously been shown to have antiinflamatory acitivies. The present study assessed whether eicosanoid biosynthesis is regulated by amiprilose HCl adn whether the regulation is influenced at the early stage of arachidonate liberation from the phospholipid by phospholipase A₂ (PLA₂). Secretiion of both prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) by peritoneal macrophages and neutrophils from amiprilose HCl-treated mice was reduced with neutrophils being slightly more sensitive to the inhibitory effects. Amiprilose HCl was less effective at inhibiting PGE₂ and LTB₄ secretion that it was . Amiprilose HCl did not have a direct inhibitory effect on the PLA₂ enzyme or on secretion of the soluble form od PLA₂. In contrast, amiprilose HCl modulated the phospholipid substrate for PLA₂ as there was inhibition of label release from [1-¹⁴C]-oleic acid-labeled substrate source (i) when labeled substrate for pure PLA₂ had been preincubated with amiprilose HCl, or (ii) when labeled peritoneal cells, which had been preincubated with amiprilose HCl, were used as a substrate source either for pure PLA₂ or for their own PLA₂. Amiprilose HCl was found to bind to peritoneal cells rapidly, but transiently, with maximal binding occurring within 5 min at 37°C. Thus, amiprilose HCl was shown to be inhibitory to secretion of PGE₂ and LTB₄, at least in part, by inhibiting the availability of substrate for PLA₂.     

Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A2 activities

Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A₂ (PLA₂) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA₂ activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO₂₍₃₎ antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes.     

Lipid Metabolizing Enzyme Activities Modulated by Phospholipid Substrate Lateral Distribution

Biological membranes contain many domains enriched in phospholipid lipids and there is not yet clear explanation about how these domains can control the activity of phospholipid metabolizing enzymes. Here we used the surface dilution kinetic theory to derive general equations describing how complex substrate distributions affect the activity of enzymes following either the phospholipid binding kinetic model (which assumes that the enzyme molecules directly bind the phospholipid substrate molecules), or the surface-binding kinetic model (which assumes that the enzyme molecules bind to the membrane before binding the phospholipid substrate). Our results strongly suggest that, if the enzyme follows the phospholipid binding kinetic model, any substrate redistribution would increase the enzyme activity over than observed for a homogeneous distribution of substrate. Besides, enzymes following the surface-binding model would be independent of the substrate distribution. Given that the distribution of substrate in a population of micelles (each of them a lipid domain) should follow a Poisson law, we demonstrate that the general equations give an excellent fit to experimental data of lipases acting on micelles, providing reasonable values for kinetic parameters—without invoking special effects such as cooperative phenomena. Our theory will allow a better understanding of the cellular-metabolism control in membranes, as well as a more simple analysis of the mechanisms of membrane acting enzymes.     

Phospholipase A2 and Its Role in Brain Tissue

Abstract: Phospholipase A₂ (PLA₂) is the name for the class of lipolytic enzymes that hydrolyze the acyl group from the sn-2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of the PLA₂-catalyzed reaction can potentially act as second messengers themselves, or be further metabolized to eicosanoids, platelet-activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can potentially alter many ongoing cellular processes. The presence of PLA₂ in the central nervous system, accompanied by the relatively large quantity of potential substrate, poses an interesting dilemma as to the role PLA₂ has during both physiologic and pathologic states. Several different PLA₂ enzymes exist in brain, some of which have been partially characterized. They are classified into two subtypes, CA²⁺-dependent and Ca²⁺-independent, based on their catalytic dependence on Ca²⁺. Under physiologic conditions, PLA₂ may be involved in phospholipid turnover, membrane remodeling, exocytosis, detoxification of phospholipid peroxides, and neurotransmitter release. However, under pathological situations, increased PLA₂ activity may result in the loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and the accumulation of lipid peroxides. These processes, along with loss of ATP, may be responsible for the loss of membrane phospholipid and subsequent neuronal injury found in ischemia, spinal cord injury, and other neurodegenerative diseases. This review outlines the current knowledge of the PLA₂ found in the central nervous system and attempts to define the role of PLA₂ during both physiologic and pathologic conditions.     

Effects of the propeptide of group X secreted phospholipase A2 on substrate specificity and interfacial activity on phospholipid monolayers

Group X secreted phospholipase A₂ (GX sPLA₂) plays important physiological roles in the gastrointestinal tract, in immune and sperm cells and is involved in several types of inflammatory diseases. It is secreted either as a mature enzyme or as a mixture of proenzyme (with a basic 11 amino acid propeptide) and mature enzyme. The role of the propeptide in the repression of sPLA₂ activity has been studied extensively using liposomes and micelles as model interfaces. These substrates are however not always suitable for detecting some fine tuning of lipolytic enzymes. In the present study, the monolayer technique is used to compare PLA₂ activity of recombinant mouse GX sPLA₂ (mGX) and its pro-form (PromGX) on monomolecular films of dilauroyl-phosphatidyl-ethanolamine (DLPE), -choline (DLPC) and -glycerol (DLPG). The PLA₂ activity and substrate specificity of mGX (PE ≈ PG > PC) were found to be surface pressure-dependent. mGX displayed a high activity on DLPE and DLPG but not on DLPC monolayers up to surface pressures corresponding to the lateral pressure of biological membranes (30–35 mN/m). Overall, the propeptide impaired the enzyme activity, particularly on DLPE whatever the surface pressure. However some conditions could be found where the propeptide had little effects on the repression of PLA₂ activity. In particular, both PromGX and mGX had similar activities on DLPG at a surface pressure of 30 mN/m. These findings show that PromGX can be potentially active depending on the presentation of the substrate (i.e., lipid packing) and one cannot exclude such an activity in a physiological context. A structural model of PromGX was built to investigate how the propeptide controls the activity of GX sPLA₂. This model shows that the propeptide is located within the interfacial binding site (i-face) and could disrupt both the interfacial binding of the enzyme and the access to the active site by steric hindrance.     

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.     

Membrane simulations mimicking acidic pH reveal increased thickness and negative curvature in a bilayer consisting of lysophosphatidylcholines and free fatty acids

Phospholipids are key components of biological membranes and their lipolysis with phospholipase A₂ (PLA₂) enzymes occurs in different cellular pH environments. Since no studies are available on the effect of pH on PLA₂-modified phospholipid membranes, we performed 50-ns atomistic molecular dynamics simulations at three different pH conditions (pH 9.0, 7.5, and 5.5) using a fully PLA₂-hydrolyzed phosphatidylcholine (PC) bilayer which consists solely of lysophosphatidylcholine and free fatty acid molecules. We found that a decrease in pH results in lateral squeezing of the membrane, i.e. in decreased surface area per headgroup. Thus, at the decreased pH, the lipid hydrocarbon chains had larger S_CD order parameter values, and also enhanced membrane thickness, as seen in the electron density profiles across the membrane. From the lateral pressure profiles, we found that the values of spontaneous curvature of the two opposing monolayers became negative when the pH was decreased. At low pH, protonation of the free fatty acid headgroups reduces their mutual repulsion and accounts for the pH dependence of all the above-mentioned properties. The altered structural characteristics may significantly affect the overall surface properties of biomembranes in cellular vesicles, lipid droplets, and plasma lipoproteins, play an important role in membrane fission and fusion, and modify interactions between membrane lipids and the proteins embedded within them.     

Phospholipase A2 catalysis and lipid mediator lipidomics

Phospholipase A₂ (PLA₂) enzymes are the upstream regulators of the eicosanoid pathway liberating free arachidonic acid from the sn-2 position of membrane phospholipids. Free intracellular arachidonic acid serves as a substrate for the eicosanoid biosynthetic enzymes including cyclooxygenases, lipoxygenases, and cytochrome P450s that lead to inflammation. The Group IVA cytosolic (cPLA₂), Group VIA calcium-independent (iPLA₂), and Group V secreted (sPLA₂) are three well-characterized human enzymes that have been implicated in eicosanoid formation. In this review, we will introduce and summarize the regulation of catalytic activity and cellular localization, structural characteristics, interfacial activation and kinetics, substrate specificity, inhibitor binding and interactions, and the downstream implications for eicosanoid biosynthesis of these three important PLA₂ enzymes.     

Interactions of Platinum and Ruthenium Coordination Complexes with Pancreatic Phospholipase A2 and Phospholipids Investigated by MALDI TOF Mass Spectrometry

Phospholipase A₂ is involved in propagation of inflammatory processes and carcinogenesis through its role in phospholipid metabolism, and release of arachidonic acid and lysophospholipids. Recent findings on correlation between elevated PLA₂ activity and metastatic cancer render this enzyme an attractive target for cancer therapy. On the other hand, due to a broad range of oxidation states under physiological conditions and a high affinity for protein binding, platinum and ruthenium coordination complexes are promising candidates for PLA₂ inhibitors. In this article, we discuss the interactions of Pt and Ru coordination complexes with PLA₂ and phospholipids, as well as the application of MALDI‐TOF mass spectrometry for screening PLA₂ inhibitors. Owing to the ability of this technique to simultaneously detect and monitor changes in substrate and product concentrations, the inhibitor mechanisms of both Pt and Ru complexes with various ligands were determined.     

A robust all-atom model for LCAT generated by homology modeling

LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A₂ (PLA₂) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15–20 Å relative to PLA₂. ii) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii) A β-hairpin resembling a lipase lid separates S181 from solvent. iv) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specificity, and K128 and R147, whose mutations cause LCAT deficiency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport.     

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.     

Sphingomyelin modulates interfacial binding of Taiwan cobra phospholipase A2

The goal of the present study is to elucidate the effect of sphingomyelin on interfacial binding of Taiwan cobra phospholipase A₂ (PLA₂). Substitution of Asn-1 with Met caused a reduction in enzymatic activity and membrane-damaging activity of PLA₂ toward phospholipid vesicles, while sphingomyelin exerted an inhibitory effect on the biological activities of native and mutated PLA₂. Incorporation of sphingomyelin reduced membrane fluidity of phospholipid vesicles as evidenced by Laurdan fluorescence measurement. The results of self-quenching studies, binding of fluorescent probe, trinitrophenylation of Lys residues and fluorescence energy transfer between protein and lipid revealed that sphingomyelin altered differently membrane-bound mode of native and mutated PLA₂. Moreover, it was found that PLA₂ and N-terminally mutated PLA₂ adopted different conformation and geometrical arrangement on binding with membrane bilayer. Nevertheless, the binding affinity of PLA₂ and N-terminal mutant for phospholipid vesicles was not greatly affected by sphingomyelin. Together with the finding that mutation on N-terminus altered the gross conformation of PLA₂, our data indicate that sphingomyelin modulates the mode of membrane binding of PLA₂ at water/lipid interface, and suggest that the modulated effect of sphingomyelin depends on inherent structural elements of PLA₂.     

Partial purification and kinetic characterization of the microsomal phospholipase A2 from thermally acclimated rainbow trout (Salmo garidneri)

Summary Phospholipase A₂ (PLA₂) was extracted from liver microsomal membranes of both 5 and 20°C-acclimated rainbow trout (Salmo gairdneri), using the non-ionic detergent, Triton X-100. Further purification was achieved by precipitation with 35–65% ammonium sulfate followed by gel filtration chromatography in the presence of 0.1% Triton X-100 on Sephadex G-200. These procedures resulted in a 30-fold purification and the removal of all traces of phospholipid from the enzyme of both warm-and cold-acclimated trout. Column elution profiles were similar for both acclimation groups, yielding a molecular weight estimate for the trout liver enzyme of 73,000. Comparisons of activity levels and kinetic parameters of PLA₂ from warm-and cold-acclimated fish, indicated that compensation for temperature at nonsaturating substrate concentrations was an attribute of both the particulate (microsomal) enzyme and the lipid-free protein. Cold acclimation resulted in higher activity belowV _max due primarily to decreased apparentK _m values. These adaptations to temperature could not be attributed to the interaction of the enzyme with the membrane lipids, but were due to qualitative changes in the enzyme that resulted from acclimation. Other adaptive qualities of PLA₂, such as reducedK _m in response to acute decreases in temperature in warm-acclimated fish, were only apparent in particulate preparations, and thus were a function of the protein-lipid complex. These data suggest that an acclimation-induced increase in the activity of PLA₂ may result in the activation of a deacylation-reacylation cycle at cold temperatures.Abbreviations PAGE polyacrylamide gel electrophoresis - PC phosphatidylcholine - PLA ₂ phospholipase A₂ - SDS sodium dodecylsulfate     

Lactadherin Inhibits Secretory Phospholipase A2 Activity on Pre-Apoptotic Leukemia Cells

Secretory phospholipase A₂ (sPLA₂) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA₂ concentrations are associated with autoimmune diseases and septic shock. Many sPLA₂’s do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A₂-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA₂ plateaued at 50–60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA₂ and >70% of human secretory phospholipase A₂-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA₂.     

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.     

Elevated Phospholipase A2 Activities in Plasma Samples from Multiple Cancers

Only in recent years have phospholipase A₂ enzymes (PLA₂s) emerged as cancer targets. In this work, we report the first detection of elevated PLA₂ activities in plasma from patients with colorectal, lung, pancreatic, and bladder cancers as compared to healthy controls. Independent sets of clinical plasma samples were obtained from two different sites. The first set was from patients with colorectal cancer (CRC; n = 38) and healthy controls (n = 77). The second set was from patients with lung (n = 95), bladder (n = 31), or pancreatic cancers (n = 38), and healthy controls (n = 79). PLA₂ activities were analyzed by a validated quantitative fluorescent assay method and subtype PLA₂ activities were defined in the presence of selective inhibitors. The natural PLA₂ activity, as well as each subtype of PLA₂ activity was elevated in each cancer group as compared to healthy controls. PLA₂ activities were increased in late stage vs. early stage cases in CRC. PLA₂ activities were not influenced by sex, smoking, alcohol consumption, or body-mass index (BMI). Samples from the two independent sites confirmed the results. Plasma PLA₂ activities had approximately 70% specificity and sensitivity to detect cancer. The marker and targeting values of PLA₂ activity have been suggested.     

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.     

Snake inhibitors of phospholipase A2 enzymes

Phospholipase A₂ (PLA₂) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA₂s and they are nontoxic. In comparison, venom PLA₂s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA₂ inhibitors (PLIα), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA₂s and appeared to be restricted to the serum of this snake family. Analysis of PLA₂ binding to recombinant fragments of PLIα indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIβ, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIγ, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIγ inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA₂s, and bee venom group III PLA₂. However, differences in the binding affinities indicated specificity for particular PLA₂s. A different representation has emerged for crotalid and viperid snakes. Their PLIγs did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the γ-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid β-neurotoxins and did not inhibit β-neurotoxins from elapids [1]. Further studies are required to determine if this phenomenon is true for all γ-type inhibitors from Crotalidae snakes. The relative distribution of these inhibitors, their specificities and the structural features involved in binding are discussed in this review.     

Differential mode of attack on membrane phospholipids by an acidic phospholipase A2 (RVVA-PLA2-I) from Daboia russelli venom

An acidic phospholipase A₂ (RVVA-PLA₂-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA₂-I was non‐lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA₂ enzyme. RVVA-PLA₂-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA₂-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24 h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA₂-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA₂s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA₂-I suggested the correlation between catalytic and membrane damaging activities of this PLA₂ enzyme. Our study advocates that the presence of a large number of PLA₂-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA₂ enzyme.