Ginseng-derived patatin-related phospholipase PgpPLAIIIß alters plant growth and lignification of xylem in hybrid poplars.

Patatin-liked phospholipase A (pPLAs) are major lipid acyl hydrolases that participate in various biological functions in plant growth and development. Previously, a ginseng-derived pPLAIII homolog was reported to reduce lignin content in Arabidopsis. This led us to evaluate its possible usefulness as a biomass source in wood plant. Herein, we report that there are six members in the pPLAIII gene family in poplar. Overexpression of pPLAIIIß derived from ginseng resulted in a reduced plant height with radially expanded stem growth in hybrid poplars. Compared with the wild type (WT), the chlorophyll content was increased in the overexpression poplar lines, whereas the leaf size was smaller. The secondary cell wall structure in overexpression lines was also altered, exhibiting reduced lignification in the xylem. Two transcription factors, MYB92 and MYB152, which control lignin biosynthesis, were downregulated in the overexpression lines. The middle xylem of the overexpression line showed heavy thickening, making it thicker than the other xylem parts and the WT xylem, which rather could have been contributed by the presence of more cellulose in the selected surface area. Taken together, the results suggest that PgpPLAIIIß plays a role not only in cell elongation patterns, but also in determining the secondary cell wall composition.

A Continuous and Sensitive Spectrophotometric Assay for Lipase and Phospholipase A Activities Using α-Eleostearic Acid-Containing Substrates.

To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen lipase and phospholipase A (PLA) activities. Here, a new ultraviolet spectrophotometric assay for lipase or PLA was developed using natural triglycerides or synthetic glycerophosphatidylcholines containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and consequently confers strong UV absorption properties of this free fatty acid as well as of lipid substrates harboring it. The substrate was coated into the wells of a microplate, and the lipolytic activities were measured by the absorbance increase at 272 nm due to the transition of α-eleostearic acid moiety from the adsorbed to the soluble state. This continuous assay is compatible with a high-throughput screening method and can be applied specifically to the screening of new potential lipase, PLA1 and PLA2 inhibitors.

Identification of a novel and potent inhibitor of phospholipase A(2) in a medicinal plant: crystal structure at 1.93Å and Surface Plasmon Resonance analysis of phospholipase A(2) complexed with berberine.

Crystal of Russell Viper venom phospholipase A(2) complexed with an isoquinoline alkaloid, berberine from a herbaceous plant Cardiospermum halicacabum, was prepared and its structure was solved by X-ray crystallography. The crystal diffracted up to 1.93Å and the structure solution clearly located the position of berberine in the active site of the enzyme. Two hydrogen bonds, one direct and the other water mediated, were formed between berberine and the enzyme. Gly 30 and His 48 made these two hydrogen bonds. Additionally, the hydrophobic surface of berberine made a number of hydrophobic contacts with side chains of neighboring amino acids. Surface Plasmon Resonance studies revealed strong binding affinity between berberine and phospholipase A(2). Enzyme inhibition studies proved that berberine is a competitive inhibitor of phospholipase A(2). It was inferred that the isoquinoline alkaloid, berberine, is a potent natural inhibitor of phospholipaseA(2).

Crystallization and preliminary X-ray diffraction analysis of a Lys49-phospholipase A2 complexed with caffeic acid, a molecule with inhibitory properties against snake venoms.

Phospholipases A(2) (PLA(2)s) are one of the main components of bothropic venoms; in addition to their phospholipid hydrolysis action, they are involved in a wide spectrum of pharmacological activities, including neurotoxicity, myotoxicity and cardiotoxicity. Caffeic acid is an inhibitor that is present in several plants and is employed for the treatment of ophidian envenomations in the folk medicine of many developing countries; as bothropic snake bites are not efficiently neutralized by conventional serum therapy, it may be useful as an antivenom. In this work, the cocrystallization and preliminary X-ray diffraction analysis of the Lys49-PLA(2) piratoxin I from Bothrops pirajai venom in the presence of the inhibitor caffeic acid (CA) are reported. The crystals diffracted X-rays to 1.65 Šresolution and the structure was solved by molecular-replacement techniques. The electron-density map unambiguously indicated the presence of three CA molecules that interact with the C-terminus of the protein. This is the first time a ligand has been observed bound to this region and is in agreement with various experiments previously reported in the literature.

Crystal structure of a heterodimer of phospholipase A2 from Naja naja sagittifera at 2.3 A resolution reveals the presence of a new PLA2-like protein with a novel cys 32-Cys 49 disulphide bridge with a bound sugar at the substrate-binding site.

The crystal structure of the phospholipase A2 (PLA2) heterodimer from Naja naja sagittifera reveals the presence of a new PLA2-like protein with eight disulphide bridges. The heterodimer is formed between a commonly observed group I PLA2 having seven characteristic disulfide bonds and a novel PLA2-like protein (Cys-PLA2) containing two extra cysteines at two highly conserved sites (positions 32 and 49) of structural and functional importance. The crystals of the heterodimer belong to tetragonal space group P41212 with cell dimensions, a = b = 77.7 A and c = 68.4 A corresponding to a solvent content of 33%, which is one of the lowest values observed so far in the PLA2 crystals. The structure has been solved with molecular replacement method and refined to a final R value of 21.6% [Rfree = 25.6%]. The electron density revealed the presence of cysteines 32 and 49 that are covalently linked to give rise to an eighth disulphide bridge in the PLA2-like monomer. A non-protein high-quality electron density was also observed at the substrate-binding site in the PLA2-like protein that has been interpreted as N-acetylglucosamine. The overall tertiary folds of the two monomers are similar having all features of PLA2-type folding. A zinc ion is detected at the interface of the heterodimer with fivefold coordination while another zinc ion was found on the surface of Cys-PLA2 with sixfold coordination. The conformations of the calcium-binding loops of both monomers are significantly different from each other as well as from those in other group I PLA2s. The N-acetylglucosamine molecule is favorably placed in the substrate-binding site of Cys-PLA2 and forms five hydrogen bonds and several van der Waals interactions with protein atoms, thus indicating a strong affinity. It also provides clue of the possible mechanism of sugar recognition by PLA2 and PLA2-like proteins. The formation of heterodimer seems to have been induced by zinc ion.

Effects of morin on snake venom phospholipase A2 (PLA2).

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA2 isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to study the effect of flavonoids on PLA2. We observed that a treatment of PLA2 with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA2 possess a second pharmacological site which does not affect or depend on the enzymatic activity.

Characterization of a cDNA encoding Arabidopsis secretory phospholipase A2-alpha, an enzyme that generates bioactive lysophospholipids and free fatty acids.

Phospholipase A2s (PLA2s) are enzymes that liberate lysophospholipids and free fatty acids (FFAs) from membrane phospholipids in response to hormones and other external stimuli. This report describes the cloning and functional characterization of a PLA2 cDNA from Arabidopsis thaliana, AtsPLA2-alpha, which represents one of four secretory PLA2 (sPLA2) genes in Arabidopsis. The encoded protein is 148-amino acid polypeptide and is predicted to contain a 20-amino acid signal peptide at its amino terminus. The predicted mature form (Mr=14,169) of AtsPLA2-alpha exhibited approximately 5 times the specific activity of its pre-processed form. Different from animal sPLA2s, AtsPLA2-alpha showed a significant preference for the acyl group linoleic acid over palmitic acid in phospholipid hydrolysis. Like some animal sPLA2s, however, it has a slight preference for phosphatidylethanolamine over phosphatidylcholine as the substrate. The specific activity of AtsPLA2-alpha continuously increased as the Ca2+ concentration was increased to 10 mM, and the optimal pH range was very broad and biphasic between 6 and 11. AtsPLA2-alpha transcript was detected at low levels in roots, stems, leaves, and flowers but not in siliques.

Rosmarinic acid, a new snake venom phospholipase A2 inhibitor from Cordia verbenacea (Boraginaceae): antiserum action potentiation and molecular interaction.

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. The methanolic extract from Cordia verbenacea (Cv) significantly inhibited paw edema induced by Bothrops jararacussu snake venom and by its main basic phospholipase A2 homologs, namely bothropstoxins I and II (BthTXs). The active component was isolated by chromatography on Sephadex LH-20 and by RP-HPLC on a C18 column and identified as rosmarinic acid (Cv-RA). Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid [2-O-cafeoil-3-(3,4-di-hydroxy-phenyl)-R-lactic acid]. This is the first report of RA in the species C. verbenacea ('baleeira', 'whaler') and of its anti-inflammatory and antimyotoxic properties against snake venoms and isolated toxins. RA inhibited the edema and myotoxic activity induced by the basic PLA2s BthTX-I and BthTX-II. It was, however, less efficient to inhibit the PLA2 activity of BthTX-II and, still less, the PLA2 and edema-inducing activities of the acidic isoform BthA-I-PLA2 from the same venom, showing therefore a higher inhibitory activity upon basic PLA2s. RA also inhibited most of the myotoxic and partially the edema-inducing effects of both basic PLA2s, thus reinforcing the idea of dissociation between the catalytic and pharmacological domains. The pure compound potentiated the ability of the commercial equine polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA2s in experimental models. CD data presented here suggest that, after binding, no significant conformation changes occur either in the Cv-RA or in the target PLA2. A possible model for the interaction of rosmarinic acid with Lys49-PLA2 BthTX-I is proposed.

Crystal structure of the complex formed between a group I phospholipase A2 and a naturally occurring fatty acid at 2.7 A resolution.

This is the first evidence of a naturally bound fatty acid to a group I Phospholipase A(2) (PLA(2)) and also to a PLA(2) with Asp 49. The fatty acid identified as n-tridecanoic acid is observed at the substrate recognition site of PLA(2) hydrophobic channel. The complex was isolated from the venom of Bungarus caeruleus (Common Indian Krait). The primary sequence of the PLA(2) was determined using the cDNA method. Three-dimensional structure has been solved by the molecular replacement method and refined using the CNS package to a final R factor of 19.8% for the data in the resolution range from 20.0 to 2.7 A. The final refined model is comprised of 912 protein atoms, one sodium ion, one molecule of n-tridecanoic acid, and 60 water molecules. The sodium ion is located in the calcium-binding loop with a sevenfold coordination. A characteristic extra electron density was observed in the hydrophobic channel of the enzyme, into which a molecule of n-tridecanoic acid was clearly fitted. The MALDI-TOF measurements of the crystals had earlier indicated an increase in the molecular mass of PLA(2) by 212 Da over the native PLA(2). A major part of the ligand fits well in the binding pocket and interacts directly with His 48 and Asp 49. Although the overall structure of PLA(2) in the present complex is similar to the native structure reported earlier, it differs significantly in the folding of its calcium-binding loop.

FlexX-Scan: fast, structure-based virtual screening.

We present a new software module, FlexX-Scan, for high-throughput, structure-based virtual screening. FlexX-Scan was developed with the aim to further speed up the virtual screening process. Based on the incremental construction docking tool FlexX (Rarey et al., J Mol Biol 1996;261:470-489), a compact descriptor for representing favorable protein interaction spots within the protein binding site has been developed. The descriptor is calculated using special-purpose clustering techniques applied to the usual interaction points created by FlexX. The algorithm automatically detects a small set of interaction spots in the binding site for positioning ligand functional groups. The parametrizations of the base placement and incremental construction algorithms have been adapted to the new interaction model. We tested the software tool on a diverse set of 200 protein-ligand complexes from the protein database (PDB) (Kramer et al., Proteins 1999;37:228-241). On average, the algorithm proposes about 90 interaction spots per binding site compared to about 1000 interaction dots in FlexX. We observe that the docking solutions of FlexX-Scan have a root-mean-square deviation from the crystal structure similar to the deviation of docking solutions of standard FlexX. For further validation we also performed virtual screening experiments for cyclin-dependent kinase 2, thrombin, angiotensin-converting enzyme, and dihydrofolat reductase. In these experiments, we screened a set of 34,000 random compounds and a number of known actives for each target. With FlexX-Scan, we achieved comparable enrichments to standard FlexX, with an averaged computing time of 5-10 s per compound, depending on parametrization.

Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.

Genetic knockout of hormone-sensitive lipase in mice has implicated the presence of other intracellular triacylglycerol (TAG) lipases mediating TAG hydrolysis in adipocytes. Despite intense interest in these TAG lipases, their molecular identities thus far are largely unknown. Sequence data base searches for proteins containing calcium-independent phospholipase A2 (iPLA2) dual signature nucleotide ((G/A)XGXXG) and lipase (GXSXG) consensus sequence motifs identified a novel subfamily of three putative iPLA2/lipase family members designated iPLA2epsilon, iPLA2zeta, and iPLA2eta (previously named adiponutrin, TTS-2.2, and GS2, respectively) of previously unknown catalytic function. Herein we describe the cloning, heterologous expression, and affinity purification of the three human isoforms of this iPLA2 subfamily in Sf9 cells, and we demonstrate that each possesses abundant TAG lipase activity. Moreover, iPLA2epsilon, iPLA2zeta, and iPLA2eta also possess acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. (E)-6-(Bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, a mechanism-based suicide substrate inhibitor of all known iPLA2s, inhibits the triglyceride lipase activity of each of the three isoforms similarly (IC50=0.1-0.5 microm). Quantitative PCR revealed dramatically increased expression of iPLA2epsilon and iPLA2zeta transcripts during the hormone-induced differentiation of 3T3-L1 cells into adipocytes and identified the presence of all three iPLA2 isoforms in human SW872 liposarcoma cells. Collectively, these results identify three novel TAG lipases/acylglycerol transacylases that likely participate in TAG hydrolysis and the acyl-CoA independent transacylation of acylglycerols, thereby facilitating energy mobilization and storage in adipocytes.

Lysosomal phospholipase A2 is selectively expressed in alveolar macrophages.

Lung surfactant is the surface-active agent comprised of phospholipids and proteins that lines pulmonary alveoli. Surfactant stabilizes the alveolar volume by reducing surface tension. Previously, we identified a lysosomal phospholipase A2, termed LPLA2, with specificity toward phosphatidylcholine and phosphatidylethanolamine. The phospholipase is localized to lysosomes, is calcium-independent, has an acidic pH optimum, and transacylates ceramide. Here, we demonstrate that LPLA2 is selectively expressed in alveolar macrophages but not in peritoneal macrophages, peripheral blood monocytes, or other tissues. Other macrophage-associated phospholipase A2s do not show a comparable distribution. LPLA2 is of high specific activity and recognizes disaturated phosphatidylcholine as a substrate. The lysosomal phospholipase A2 activity is six times lower in alveolar macrophages from mice with a targeted deletion of the granulocyte macrophage colony-stimulating factor (GM-CSF), a model of impaired surfactant catabolism, compared with those from wild-type mice. However, LPLA2 activity and protein levels are measured in GM-CSF null mice in which GM-CSF is expressed as a transgene under the control of the surfactant protein C promoter. Thus LPLA2 may be a major enzyme of pulmonary surfactant phospholipid degradation by alveolar macrophages and may be deficient in disorders of surfactant metabolism.

Phaiodactylipin, a glycosylated heterodimeric phospholipase A from the venom of the scorpion Anuroctonus phaiodactylus.

Phaiodactylipin was purified from the venom of the scorpion Anuroctonus phaiodactylus. It is the first protein to be purified from a scorpion of the family Iuridae and has a molecular mass of 19 172 atomic mass units. The mature protein is composed of two subunits, the large one consisting of 108 amino acid residues, whereas the small subunit has only 18 residues, and the structure is stabilized by five disulfide bridges. The heterodimer is expressed from a single message containing 769 base pairs and a signal peptide with 16 and/or 25 amino acid residues. During maturation an internal hexapeptide is excised. There are three putative sites of N-glycosylation, one of which is situated in the small subunit region. The carbohydrate composition of this site was determined by mass spectrometry analysis and was found to contain three hexoses, two N-acetyl-hexoses and two deoxyhexoses. The protein has a calcium dependent phospholipase A(2) type of activity. It is lethal to arthropods (insects and isopods), but not toxic to mammals, using doses up to 20 microg per 20 g mouse body weight. For crickets, a dose of 5 microg per animal is lethal; however, when injected into mice it is capable of causing only muscular inflammation, without rupture of the basal membrane of cells. It has a direct hemolytic effect in human erythrocytes and retards the coagulation time of blood. It is an unusual phospholipase A(2), with only 36% and 50% amino acid sequence identities to the closest known phospholipases, imperatoxin I and phospholipin, respectively. Identities with bee and Heloderma venom phospholipase are only in the order of 28%.

Crystal structures of BnSP-7 and BnSP-6, two Lys49-phospholipases A(2): quaternary structure and inhibition mechanism insights.

Phospholipases A(2) are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. A class of PLA(2)-like proteins has been described which despite PLA(2) activity on artificial substrate, due to a D49K mutation, is still highly myonecrotic. This work reports the X-ray structure determination of two Lys49-PLA(2)s from Bothrops neuwiedi pauloensis (BnSP-7 and BnSP-6) and, for the first time, the comparison of eight dimeric Lys49-PLA(2)s. This comparison reveals that there are not just two ("open" and "closed") but at least six different conformations. The binding of fatty acid observed in three recent Lys49-PLA(2) structures seems to be independent of their quaternary conformation. Cys29 polarization by Lys122 is not significant for BnSP-7 and BnSP-6 or other structures not bound by fatty acids. These structures may be in an "active" state when nothing is bound to them and the Lys122/Cys29 interactions are weak or absent.

Characterization of Arabidopsis secretory phospholipase A2-gamma cDNA and its enzymatic properties.

Plant secretory phospholipases A(2) (sPLA(2)s) probably play important roles in phospholipid signaling based on the data reported from other organisms, but their functions are poorly understood because of the lack of cloned sPLA(2) genes. In this study, we cloned and characterized an Arabidopsis secretory phospholipase A(2)-gamma (AtsPLA(2)-gamma) cDNA, and examined its enzymatic properties. The recombinant protein of AtsPLA(2)-gamma showed maximal enzyme activity at pH 8.0, and required Ca(2+) for activity. Moreover, AtsPLA(2)-gamma showed sn-2 position specificity but no prominent acyl preference, though it showed head group specificity to phosphatidylethanolamine rather than to phosphatidylcholine. AtsPLA(2)-gamma was found to predominate in the mature flower rather than in other tissues, and subcellular localization analysis confirmed that AtsPLA(2)-gamma is secreted into the intercellular space.

Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay.

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported to serve as a storage protein and also to exhibit lipid phospholipase activity. This paper describes a simple continuous spectrophotometric method for assaying patatin phospholipase activity. The procedure is based on a coupled enzymatic assay using [1,2-dilinoleoyl] PC as the phospholipase substrate and lipoxygenase as the coupling enzyme. In the procedure developed in this work, lipoxygenase oxidizes the linoleic acid released by the phospholipase activity of patatin. This activity can then be followed spectrophotometrically by recording the increase in absorbance at 234 nm that results from the formation of the corresponding hydroperoxide from linoleic acid by the action of lipoxygenase. The optimal assay concentrations of patatin and lipoxygenase were established. Phospholipase activity varied with pH, reaching its optimal value at pH 9.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (Vm = 9.8 x 10(-3) micromol/min x microg protein, Km = 7.8 microM, Vm/Km = 1.3 min(-1) x microg protein). This method proved to be specific since there was no activity in the absence of patatin. It also had the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase.

Arachidonate release and prostaglandin production by group IVC phospholipase A2 (cytosolic phospholipase A2gamma).

While the role of the group IVA Ca(2+)-dependent cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in arachidonic acid (AA) metabolism has been well documented, that of its paralogue, Ca(2+)-independent group IVC PLA(2) (cPLA(2)gamma), has remained uncertain. Here we show, using a transfection strategy, that cPLA(2)gamma has the ability to increase the spontaneous and stimulus-induced release of cellular fatty acids. The AA released by cPLA(2)gamma was metabolized further to prostaglandin E(2) via cyclo-oxygenase-1 (COX-1) in the immediate response, and via COX-2 in the delayed response. Mutation of the putative catalytic-centre residue Ser(82) abrogated the AA-releasing function of cPLA(2)gamma both in vitro and in vivo. Confocal microscopy revealed that cPLA(2)gamma was distributed in the perinuclear endoplasmic reticulum membranes. Mutating the C-terminal prenylation site of cPLA(2)gamma abrogated its intracellular membrane localization and cellular AA-releasing function, without reducing its enzyme activity in vitro. Our results indicate that cPLA(2)gamma is the second cPLA(2) enzyme that contributes to cellular AA metabolism and phospholipid remodelling under appropriate conditions.

Anti-inflammatory activity of Chinese medicinal vine plants.

Anti-inflammatory activities of ethanol extracts from nine vine plants used in traditional Chinese medicine to treat inflammatory conditions were evaluated against a panel of key enzymes relating to inflammation. The enzymes included cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), phospholipase A(2) (PLA(2)), 5-lipoxygenase (5-LO) and 12-lipoxygenase (12-LO). The vine plants studied were: the stem of Spatholobus suberectus Dunn, the stem of Trachelospermum jasminoides Lem., the root from Tripterygium wilfordii Hook. f., the stem of Sinomenium acutum Rehder and Wilson, the stem of Piper kadsura (Choisy) Ohwi, the stem of Polygonum multiflorum Thunb., the root and stem from Tinospora sagittata Gagnep., the root of Tinospora sinensis (Lour.) Merrill, and the stem of Clematis chinensis Osbeck. All of the plant extracts showed inhibitory activities against at least one of the enzymes in various percentages depending upon the concentrations. The extract from S. suberectus was found to be active against all enzymes except COX-2. Its IC(50) values were 158, 54, 31 and 35 microg/ml in COX-1, PLA(2), 5-LO and 12-LO assays, respectively. T. jasminoides showed potent inhibitory activities against both COX-1 (IC(50) 35 microg/ml) and PLA(2) (IC(50) 33 microg/ml). The most potent COX-1, COX-2 and 5-LO inhibition was observed in the extract of T. wilfordii with the IC(50) values of 27, 125 and 22 microg/ml, respectively. The findings of this study may partly explain the use of these vine plants in traditional Chinese medicine for the treatment of inflammatory conditions.

Positional distribution of FA in TAG of enzymatically modified borage and evening primrose oils.

Stereospecific analysis was carried out to establish positional distribution of FA in the TAG of DHA, EPA, and (EPA + DHA)-enriched oils. In this study, TAG of enzymatically modified oils were purified using a silicic acid column. The TAG were then subjected to positional distribution analysis using a modified procedure involving reductive cleavage with Grignard reagent. The results showed that in DHA-enriched borage oil (BO), DHA was randomly distributed over the three positions of TAG, whereas gamma-linolenic acid (GLA) was mainly esterified at the sn-2 and -3 positions. In DHA-enriched evening primrose oil (EPO), however, DHA and GLA were concentrated in the sn-2 position. In EPA-enriched BO, EPA was randomly distributed over the three positions of TAG, similar to that observed for DHA. In EPA-enriched EPO, however, this FA was mainly located at the primary positions (sn-1 and sn-3) of TAG. In both oils, GLA was preferentially esterified at the sn-2 position. In (EPA + DHA)-enriched BO, EPA and DHA were mainly esterified at the sn-1 and -3 positions of TAG, whereas GLA was mainly located at the sn-2 position. In (EPA + DHA)-enriched EPO, GLA was mainly located at the sn-2 and -3 positions; EPA was preferentially esterified at the sn-1 and -3 positions, and DHA was found mainly at the sn-3 position.

Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2.

Bothropstoxin-I (BthTx-I) is a Lys(49)-phospholipase A(2) from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca(2+)-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117-122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys(122), these same substitutions did not significantly alter the Ca(2+)-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca(2+)-independent membrane-damaging activity but, with the exception of Lys(122), had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115-119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A(2), and support a model of a Ca(2+)-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.