Characterization of three edema-inducing phospholipase A2 enzymes from habu (Trimeresurus flavoviridis) venom and their interaction with the alkaloid aristolochic acid

A basic phospholipase A2 (PLA2) enzyme, TFV PL-X (pI 9.2) and two acidic PLA2 enzymes, TFV PL-Ia (pI 4.9) and TFV PL-Ib (pI 4.5) were purified from Trimeresurus flavoviridis venom on CM-Sephadex C-25 and QAE-Sephadex A-25 columns, respectively. The basic enzyme exists as a monomer, whereas the acidic enzymes are dimers. These enzymes differ in properties such as molecular weight, Km, optimum pH and temperature and pharmacological properties. The basic enzyme hydrolysed purified phospholipids in the order of PC greater than PE greater than PS greater than PI = 0, while for TFV PL-Ia and TFV PL-Ib the order was PC greater than PE greater than PS = PI = 0. TFV PL-X was comparatively more toxic, with an LD50 value of 4.2 micrograms/g (i.p.), while the acidic PLA2 enzymes had LD50 values above 8 micrograms/g (i.p.). All three enzymes induced edema when injected into the mouse foot pad. Aristolochic acid, an alkaloid (8-methoxy-6-nitrophenanthro(3,4-d)-1,3-dioxole-5-carboxylic acid) from the medicinal plant Aristolochia radix, interacts with these PLA2 enzymes. It is a competitive inhibitor with varying affinity when PC is used as substrate. Aristolochic acid inhibits direct and indirect hemolytic activity, as well as edema-inducing activity, of TFV PL-X, but fails to neutralize the lethal potency of the enzyme. On the other hand, it inhibits direct and indirect lytic activity of TFV PL-Ia and TFV PL-Ib only at 10-fold higher concentrations and it enhances the edema-inducing activity of these enzymes. Such effects of aristolochic acid indicates that (1) different mechanisms may be involved in the edema-inducing activity of PLA2 enzymes and (2) catalytic and pharmacological sites are separate on the PLA2 molecule.     

Purification and characterization of a neurotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom.

Snake venoms contain multimolecular forms of phospholipase A2 which are diverse with respect to their pharmacological properties. A neurotoxic PLA2 from Naja naja naja venom has been purified in two steps. (1) The whole venom was fractionated on CM-Sephadex C-25 column; 4.6% of the total PLA2 activity recovered was found in the NN-V fraction. (2) The NN-Vb-PLA2 fraction was purified to homogeneity by gel filtration of fraction NN-V on Sephadex G-50. It is a basic protein with a mol. wt between 10,500 and 11,000, and is more toxic than other basic PLA2s purified from Naja naja naja venom. The LD50 of NN-Vb-PLA2 is 0.27 mg/kg body wt. It induced neurotoxic symptoms in experimental mice and is devoid of myotoxic, anticoagulant and edema-inducing activities.     

Characterization, amino acid sequence and evolution of edema-inducing, basic phospholipase A2 from Trimeresurus flavoviridis venom.

Two phospholipases A2 (PLA2s) were purified from the venom of Trimeresurus flavoviridis (Crotalinae) inhabiting Tokunoshima island, Japan, and named PLA-A and PLA-B in the order of elution on a cation-exchange column. Lipolytic activities of PLA-A and PLA-B toward mixed micelles and liposomes were substantially lower than that of PLA2 (an [Asp49]PLA2) which had been isolated from the same venom. Both PLA-A and PLA-B consisted of 122 amino acids and contained aspartate at position 49 (the numbering according to the aligned sequences of PLA2s in Fig. 8), thus belonging to an [Asp49]PLA2 subgroup. PLA-A and PLA-B were identical in sequence with an exception at position 79. PLA-B contained Asn-Gly at positions 79 and 80 which are located in the beta-sheet region. On the other hand, PLA-A had beta-Asp-Gly and alpha-Asp-Gly in high and low proportion, respectively, at the corresponding positions which were produced from Asn-Gly through the base-catalyzed formation and hydrolysis of the succinimide type intermediate. Thus, PLA-A is derived from PLA-B. PLA-B is similar in sequence to PL-X, which had been purified from the venom of T. flavoviridis inhabiting Amami-Oshima island, Japan, and to PL-X', whose cDNA had been cloned from Tokunoshima T. flavoviridis venom gland, rather than PLA2. PLA-B showed strong edema-inducing activity, while PLA-A exhibited rather lower activity. The sequence around position 79 which constitutes a beta-turn segment seems to be crucial for edema-inducing activity. Phylogenetic tree of Tokunoshima T. flavoviridis venom PLA2 isozymes indicated that PLA-B and PL-X' diverged from PLA2 after branching of [Asp49]PLA2 forms and [Lys49]PLA2 forms.     

Koninginins, phospholipase A2 inhibitors from endophytic fungus Trichoderma koningii.

Many isolated compounds from endophytic fungus have been useful to human beings, mainly those with medicinal applications and particularly those that can be used in inflammatory processes. Trichoderma fungi produce substances known as koninginins that have great structural similarity to compounds like flavonoids and vitamin E, which are able to inhibit the phospholipase A(2) (PLA(2)). In this work, koninginins A, E and F (KonA, KonE and KonF, respectivamente) isolated from Trichoderma koningii had their capabilities of inhibiting edema-inducing, myotoxic and enzymatic activities of the total venom of Bothrops jararacussu (jararacu?u) snake analyzed, as well as one of its homolog forms of phospholipases A(2) (bjPLA(2)-group IIB) and human secreted PLA(2) protein fusion (hsPLA(2)-group IIA). KonA was not efficient in inhibiting the three activities analyzed in all the tests performed. Nevertheless, KonE and KonF present great capability in inhibiting the effects provoked not only by the venom but also by both PLA(2). The activities inhibition shown by KonE and KonF over the enzymes is significantly higher than those obtained over the total venom. KonE and KonF were slightly more efficient in the inhibition of the group IIB (bjPLA(2)) PLA(2) effects than in the inhibition of the group IIA (hsPLA(2)) PLA(2) effects. KonE and KonF structures are similar to vitamin E and, possibly, the action mode of these molecules is similar to the one produced by the vitamin. These results, apparently, indicate that koninginins E and F, as well as vitamin E, present structural regions that might be used as start points in seeking for new and specific anti-inflammatory drugs against such enzymes.     

Three toxins with phospholipase activity isolated from the yellow-legged hornet (Vespa verutina) venom.

The yellow-legged hornet, Vespa verutina, is widely distributed in both the mountain area and the suburbs of Taiwan and possesses highly toxic venom (LD50=0.02 microl/g mouse). By gel filtration on Fractogel (TSK HW 50f) followed by cation-exchange chromatography on Mono S column, three toxins designated as verutoxin 1, 2a and 2b (VT-1, VT-2a and VT-2b) were isolated from the venom. The toxin VT-1 had a molecular mass of 34,982 Da and an LD50 value of 3.61 microg/g mouse. Toxin VT-2a and 2b were more basic and more toxic than VT-1. VT-2a and 2b were isotoxins with molecular masses differing in only 14 Da (33,360 and 33,374 Da, respectively) and had a similar toxicity in mice (LD50=0.87 microg/g mouse). All three toxins were capable of catalyzing the hydrolysis of emulsified phospholipids and lysophosphatide, but not sphingomyelin. Analysis of the hydrolyzed products (fatty acid and lyso-compound) by a liquid chromatography/mass spectrometer revealed that the toxins liberates fatty acid mainly from the 1-position of the synthetic phospholipid. This result indicates that verutoxins possess phospholipase A1 activity. Toxin VT-1 showed higher phospholipase activity than VT-2a and 2b. However, the latter toxins exhibited much higher direct hemolytic activity toward the mouse red blood cells. Vespid phospholipases are known as one of the three major venom allergens in many species of wasps. Our studies indicate that vespid phospholipases A1, in addition to acting as allergens, possess direct toxic actions that may also cause death in animals. Toxin VT-2a and 2b which possess potent hemolytic activity and high lethality in mice may act as the lethal factor of V. verutina venom.     

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.     

Comparative characterization of two toxic phospholipases A2 from Indian cobra (Naja naja naja) venom.

Indian cobra venom contains many phospholipase A2 (PLA2) toxins. In the present study two toxic PLA2s have been purified from the Indian cobra (Naja naja naja) venom by column chromatography. The NN-XIa-and NN-XIb-PLA2s have mol. wts between 10,700 and 15,000. The NN-XIa-PLA2 induces myotoxic effects, oedema and neurotoxicity in mice and has an i.p. LD50 of 8.5 mg/kg body weight. The NN-XIa-PLA2 is also cytotoxic to Ehrlich ascites tumour cells. The other PLA2, NN-XIb, in contrast has an i.p. LD50 of 0.22 mg/kg body weight, and it induces acute neurotoxicity. The NN-XIb-PLA2 is devoid of the other biological activities which are exhibited by NN-XIa-PLA2.     

Biochemical and enzymatic changes after black scorpion Heterometrus fastigiousus Couzijn envenomation in experimental albino mice

The toxic effects of Asian black scorpion Heterometrus fastigiousus (Family, Scorpionidae) venom were determined in albino mice (NIH strain). Venom was isolated and fractioned by Sepharose CL-6B column chromatography. The toxicity of fractioned venom was determined in albino mice by subcutaneous envenomation. The LD(50) of venom was found to be 15 mg kg(-1) body weight and range of molecular weight of venom proteins responsible for toxicity was found from 9.5-63 kDs. The effects of fractioned venom on different biochemical and enzymatic parameters in blood serum and gastrocnemius muscle tissue of albino mice were determined after experimental envenomation. An increase in serum levels of glucose, free amino acids, uric acid, pyruvic acid and total protein was observed while a decrease in the cholesterol level in serum was observed after 4 h of envenomation. Increase in alkaline phosphatase (ALP), acid phosphatase (ACP), lactic dehydrogenase (LDH) and glutamate-pyruvate transaminase (GPT) enzyme activity in serum was observed. Glycogen content in liver, atria, ventricle, rectus abdominus and gastrocnemius muscle was decreased after experimental envenomation. Activity of ALP, ACP, LDH, GPT, AChE and Na+K+ATPase enzymes in gastrocnemius muscle tissue of envenomed albino mice was studied. Inhibition in ALP, AChE and Na+K+ATPase enzyme activity and increase in ACP, LDH and GPT enzyme activity was observed in gastrocnemius muscle after scorpion envenomation. In vitro studies with AChE and Na+K+ATPase enzymes indicated that enzymatic activity of AChE was inhibited competitively by fractioned venom in gastrocnemius muscle.     

Purification and characterization of a major phospholipase A2 from Russell's viper (Vipera russelli) venom

A major phospholipase A2 (VRV PL-VIIIa) which constitutes 24% of the whole Vipera russelli venom was purified to homogeneity by CM-Sephadex C-25 column chromatography followed by gel filtration on Sephadex G-50. VRV PL-VIIIa is a basic protein with a molecular weight of 11,800 by SDS-PAGE. This enzyme contributes 45% of the total PLA2 activity of the venom, but it is least toxic compared to other purified basic PLA2 enzymes prepared from V. russelli venom. The LD50 value (i.p.) of VRV PL-VIIIa is 5.3 mg/kg body wt. It shows neurotoxic symptoms and damages vital organs such as lung, liver and kidney at LD50 doses. It induces myonecrosis when injected i.m. into the thigh muscle of mice and edema when injected into the foot pads.     

Toxicity and mAChRs binding activity of Cassiopea xamachana venom from Puerto Rican coasts.

A separation of toxic components from the upside down jellyfish Cassiopea xamachana (Cx) was carried out to study their cytotoxic effects and examine whether these effects are combined with a binding activity to cell membrane receptors. Nematocysts containing toxins were isolated from the autolysed tentacles, ruptured by sonication, and the crude venom (CxTX) was separated from the pellets by ultracentrifugation. For identifying its bioactive components, CxTX was fractionated by gel filtration chromatography into six fractions (named fraction I-VI). The toxicity of CxTX and fractions was tested on mice; however, the hemolytic activity was tested on saline washed human erythrocytes. The LD50 of CxTX was 0.75 microg/g of mouse body and for fraction III, IV and VI were 0.28, 0.25 and 0.12 microg/g, respectively. Fractions I, II and V were not lethal at doses equivalent to LD50 1 microg/g. The hemolytic and phospholipase A2 (PLA2) activities of most fractions were well correlated with their mice toxicity. However, fraction VI, which contains the low molecular mass protein components (< or =10 kDa), has shown no PLA2 activity but highest toxicity to mice, highest hemolytic activity, and bound significantly to the acetylcholine muscarinic receptors (mAChRs) isolated from rat brain. The results suggested that fraction VI contains proteinaceous components contributing to most of cytolysis as well as membrane binding events. Meanwhile, fraction IV has shown high PLA2 that may contribute to the venom lethality and paralytic effects.     

Short Linear Motifs Characterizing Snake Venom and Mammalian Phospholipases A2

Snake venom phospholipases A2 (PLA2s) have sequences and structures very similar to those of mammalian group I and II secretory PLA2s, but they possess many toxic properties, ranging from the inhibition of coagulation to the blockage of nerve transmission, and the induction of muscle necrosis. The biological properties of these proteins are not only due to their enzymatic activity, but also to protein–protein interactions which are still unidentified. Here, we compare sequence alignments of snake venom and mammalian PLA2s, grouped according to their structure and biological activity, looking for differences that can justify their different behavior. This bioinformatics analysis has evidenced three distinct regions, two central and one C-terminal, having amino acid compositions that distinguish the different categories of PLA2s. In these regions, we identified short linear motifs (SLiMs), peptide modules involved in protein–protein interactions, conserved in mammalian and not in snake venom PLA2s, or vice versa. The different content in the SLiMs of snake venom with respect to mammalian PLA2s may result in the formation of protein membrane complexes having a toxic activity, or in the formation of complexes whose activity cannot be blocked due to the lack of switches in the toxic PLA2s, as the motif recognized by the prolyl isomerase Pin1.     

Group IIA secretory PLA2 inhibition by ursolic acid: a potent anti-inflammatory molecule

Ursolic acid (3beta-hydroxy-urs-12-en-28-oic acid) isolated from many medicinal plants has diverse pharmacologically important properties, including strong anti-inflammatory activity. However its interaction with pro-inflammatory PLA2 is not known. Ursolic acid inhibited secretory PLA2 (sPLA2) enzymes purified from Vipera russelli, Naja naja venom and human pleural fluid and synovial fluid. IC50 values determined for these enzymes ranged from 12 to 18 microM. Group II secretory PLA2 from both venoms & human inflammatory source were found to be sensitive to inhibition in comparison with group I cobra venom sPLA2. Variation in Ca2+ concentration from 2.5-15 mM did not alter the level of inhibition. Similarly sPLA2 inhibition by ursolic acid is independent of substrate concentration. Ursolic acid interacts with purified venom sPLA2 enzymes and enhances relative fluorescence intensity in a dose dependent manner. In the presence of ursolic acid apparent shift in the far UV-CD spectra of sPLA2 was observed, indicating a direct interaction with the enzyme and formation of enzyme-ursolic acid complex. This complex results in irreversible inhibition of sPLA2 as evident by dialysis study. Inhibition of sPLA2 induced mouse paw edema and indirect hemolytic activity confirmed its sPLA2 inhibitory activity in vivo and in situ respectively. These studies revealed that the strong anti-inflammatory activity of ursolic acid is by inhibiting sPLA2 enzymes.     

Structure-function relationships among neurotoxic phospholipases: NN-XIII-PLA2 from Indian cobra (Naja naja naja) and VRV PL-V from Russell's viper (Vipera russelli) venoms

Though venom phospholipases induce various pharmacological effects their mechanism of action is in some cases unclear. There may be separate pharmacological sites on the venom phospholipase molecule. In order to understand the structure-function relationships among venom phospholipases, studies on interaction of venom phospholipases with its antibodies and various alkaloids were carried out. The alkaloids aristolochic acid, ajmaline and reserpine were incapable of inhibiting the phospholipase A2 activity of NN-XIII-PLA2 but inhibited its edema inducing potency and partially inhibited the symptoms of neurotoxicity. The direct and indirect hemolytic activity remain unaffected. Polyclonal antibodies (anti PL-V Ig) to a neurotoxic PLA2 VRV PL-V neutralized the neurotoxic symptoms and lethality of VRV PL-V without affecting its in vitro phospholipase A2 activity when egg PC was used as the substrate. However, they inhibited the catalytic activity of VRV PL-V when synaptosomes were used as the substrate. Our results indicate the presence of multiple pharmacologically active sites apart from catalytic site.     

Mitochondrial dysfunction induced by pancreatic and crotalic (Crotalus durissus terrificus) phospholipases A2 on rabbit proximal tubules suspensions

In the present study we show that phospholipases A2 isolated from porcine pancreas (PP-PLA2) and Crotalus durissus terrificus snake venom (SV-PLA2) induced dose-dependent increases of LDH release from rabbit proximal tubules in suspension. Both porcine and crotalic PLA(2)s induced increases in non-esterified fatty acid (NEFA) levels (microg of NEFA/mg of tubule protein). It was observed that the NEFA levels in the pellets were higher than in the supernatant for both PLA2, and were dose-dependent for the crotalic PLA2 group. Furthermore, snake venom PLA2 induced a decrease in mitochondrial membrane potential (DeltaPsi(m)) assessed by both JC-1 uptake and safranin O uptake. Porcine PLA2 produced no effects on JC-1 uptake with the highest concentrations and an unexpected increase in the group treated with the lowest concentration. In contrast, the safranin O method revealed decreases of energization with both phospholipases, so it had higher sensitivity to the presence of the increased NEFA levels. Addition of delipidated bovine serum albumin (dBSA) completely reversed the effects induced by phospholipases on DeltaPsi(m) measured with safranin O. Incubation with pancreatic and crotalic phospholipases A2 produced no changes on cell ATP levels. We conclude that the treatment of proximal tubule suspensions with porcine or crotalic phospholipases disturbed membrane integrity as well as mitochondrial function. Specific early NEFA-mediated mitochondrial effects of the phospholipases used in the present study are indicated by the benefit provided by dBSA.     

Humanized-Single Domain Antibodies (VH/V(H)H) that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.     

Trimeresurus flavoviridis (habu snake) venom induces human erythrocyte lysis through enzymatic lipolysis, complement activation and decreased membrane expression of CD55 and CD59

Trimeresurus flavoviridis (habu snake) bites can be fatal to man because of its virulent venom, which is clinicopathologically classified as haemorrhagic, necrotic, and haemolytic toxins. Trimeresurus flavoviridis venom causes lysis of human erythrocytes in conditions where plasma is present as well as in plasma-free conditions in a dose-dependent manner. The haemolytic process requires Ca2+ and Mg2+ ions in the solution. Additionally, the venom initiates activation of the human complement cascade as evidenced by C3a and C5a releases, complement consumption indicated by CH50 and formation of soluble membrane attack complex. The insertion of membrane attack complex into the erythrocyte membranes is morphologically identified by electronmicroscopy. Immunofluorescence analysis reveals that incubation of erythrocytes with the venom decreased cell-surface expression of CD55 (decay accelerating factor) and CD59 (protectin), which renders erythrocyte more vulnerable to adherent C3 and C5 convertases and to polymerization of C9 into membranes, and may enhance autologous complement-mediated haemolysis triggered by the venom. Our data demonstrate that Trimeresurus flavoviridis venom induces haemolysis in the presence of plasma by three distinct mechanisms, direct lipolysis through PLA2 activity, activation of the human complement system, and cleavages of CD55 and CD59 from erythrocyte membranes.     

Two purified and characterized phospholipases A2 from Cerastes cerastes venom, that inhibit cancerous cell adhesion and migration

Two non-toxic PLA2s were purified to homogeneity from Cerastes cerastes Tunisian snake venom. The purification process employed gel filtration on Sephadex G-75 followed by C18 reverse phase high-pressure liquid chromatography. These two acidic enzymes, namely CC-PLA2-1 and CC-PLA2-2, have a molecular weight of 13,737.52 and 13,705.63 Da, respectively. These two PLA2 are the first reported glycosylated phospholipases A2 purified from snake venom. The rates of glycosylation are 2.5% and 0.5% (w/w), respectively. Specific activities of 1800 U/mg and 2400 U/mg for CC-PLA2-1 and CC-PLA2-2, respectively, were measured at optimal conditions. CC-PLA2-1 and CC-PLA2-2 strongly inhibited coagulation. They also exhibited a marked dose-dependent inhibitory effect on platelet aggregation induced by ADP and arachidonic acid in platelet-rich plasma. Interestingly, CC-PLA2-1 and CC-PLA2-2 inhibited in a dose-dependent manner adhesion of IGR39 melanoma and HT1080 fibrosarcoma cells to fibrinogen and fibronectin. Furthermore, both CC-PLA2-1 and CC-PLA2-2 abolished HT1080 cell migration towards fibrinogen and fibronectin. This activity is reported for the first time for PLA2 enzymes.     

Purification, sequencing and structural characterization of the phospholipase A1 from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae).

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 Da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients.     

Isolation of the major lethal toxin in the venom of Bungarus flaviceps.

The major lethal toxin in the venom of Bungarus flaviceps has been isolated by ion-exchange chromatography, absorption chromatography and RP-HPLC with a 14-fold purification and an overall yield of 16.5% of the lethal toxicity contained in crude venom. Its sublethal dose (LD(50)) determined in mice weighing 18-20 g was 0.25 (0.19-0.32) microg per mouse. The lethal toxin was pure according to disc- and SDS-PAGE as well as gel HPLC. Its apparent molecular weight determined by SDS-PAGE was 29 kDa. It is a basic protein consisting of two polypeptide chains having apparent molecular weights of 17 and 8 kDa, respectively. The toxin has PLA activity but is free of ACE activity.     

Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A2 from Agkistrodon piscivorus piscivorus snake venom

The basic Lys-49 phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus venom is homologous to the basic Asp-49 PLA2 from the same venom as well as other snake venom PLA2 enzymes. It differs, however, in several respects, most important being replacement of the previously invariant Asp-49 at the calcium binding site by Lys, resulting in a reversed order of addition of calcium and phospholipid, phospholipid binding first. Although the preferences for phospholipid substrates of the two enzymes are identical, the apparent Vmax of the Lys-49 PLA2 was only 1.4 to 3% that of the Asp-49 enzyme. Similarly, the Lys-49 PLA2, compared to the Asp-49 PLA2 had less than 3% of the intraventricular lethal potency and 4% of the anticoagulant activity. The intravenous lethal potency of the Lys-49 enzyme was 20% that of the Asp-49 PLA2 and both had little direct hemolytic activity. In contrast, both enzymes were approximately equipotent on the phrenic nerve-diaphragm preparation and on the isolated ventricle strip of the heart. On the cardiac and neuromuscular preparations, the effects of the Asp-49 PLA2 were accompanied by hydrolysis of phosphatidylcholine and phosphatidylethanolamine, whereas no phospholipid hydrolysis was observed with the Lys-49 PLA2. Evaluation of the present results, along with earlier findings using Asp-49 PLA2 enzymes from Naja nigricollis, Hemachatus haemachatus and Naja naja atra venoms, allows us to conclude that: The A. p. piscivorus Asp-49 PLA2 enzyme resembles the Asp-49 enzymes from N. n. atra and H. haemachatus. In contrast, the A. p. piscivorus Lys-49 PLA2 has much lower enzymatic and anticoagulant activities than the Asp-49 enzymes, but equal cardiotoxic and junctional effects. In contrast to some previous suggestions, basic PLA2 enzymes are not necessarily more toxic than neutral or acidic enzymes. Pharmacological effects upon the heart and phrenic nerve-diaphragm preparation correlate neither with in vitro measurements of PLA2 activity nor with actual levels of phospholipid hydrolysis in the heart or diaphragm. This suggests that PLA2 enzymes exert effects independent of phospholipid hydrolysis.