BjussuSP-I: a new thrombin-like enzyme isolated from Bothrops jararacussu snake venom.

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr=61,000 under reducing conditions and pI approximately 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated serine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca(2+) and Mg(2+)). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against 11 venom samples of Bothrops, 1 of Crotalus, and 1 of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDINEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom.

Molecular and functional characterization of a new non-hemorrhagic metalloprotease from Bothrops jararacussu snake venom with antiplatelet activity.

BjussuMP-II is an acidic low molecular weight metalloprotease (Mr approximately 24,000 and pI approximately 6.5), isolated from Bothrops jararacussu snake venom. The chromatographic profile in RP-HPLC and its N-terminal sequence confirmed its high purity level. Its complete cDNA was obtained by RT-PCR and the 615bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteases showed a high structural similarity, mainly among class P-I proteases. The molecular modeling analysis of BjussuMP-II showed also conserved structural features with other SVMPs. BjussuMP-II did not induce hemorrhage, myotoxicity and lethality, but displayed dose-dependent proteolytic activity on fibrinogen, collagen, fibrin, casein and gelatin, keeping stable at different pHs, temperatures and presence of several divalent ions. BjussuMP-II did not show any clotting or anticoagulant activity on human citrated plasma, in contrast to its inhibitory effects on platelet aggregation. The aspects broached, in this work, provide data on the relationship between structure and function, in order to better understand the effects elicited by snake venom metalloproteases.

Isolation and functional characterization of a new myotoxic acidic phospholipase A(2) from Bothrops pauloensis snake venom.

This article reports the purification procedure and the biochemical/functional characterization of Bp-PLA(2), a new myotoxic acidic phospholipase A(2) from Bothrops pauloensis snake venom. It was highly purified through three chromatographic steps (ion-exchange on CM-Sepharose, hydrophobic chromatography on Phenyl-Sepharose and RP-HPLC on a C8 column). Bp-PLA(2) is a single-chain protein of 15.8kDa and pI 4.3. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 585.3U/mg. It displayed a high indirect hemolytic activity and inhibited platelet aggregation induced by collagen or ADP. It also induced in vivo edema and myotoxicity. Pretreatment of Bp-PLA(2) with BPB reduced the enzymatic activity, the inhibitory action on platelet aggregation and myotoxicity in vitro. Morphological analyses indicated that Bp-PLA(2) induced an intense edema, with visible leukocyte infiltrate and damaged muscle cells 24h after injection. Acidic myotoxic PLA(2)s from Bothrops snake venoms are still not extensively explored and knowledge of their structural and functional features will contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.

Evaluation of three Brazilian antivenom ability to antagonize myonecrosis and hemorrhage induced by Bothrops snake venoms in a mouse model.

Despite preventing death after snakebites, there is little evidence that polyvalent antivenoms (PAVs) protect against myotoxicity and local damages. We evaluated antibothropic Brazilian PAVs from three manufacturers against the myotoxicity and hemorrhagic activity of Bothrops jararacussu and B. jararaca venoms, respectively, by using two protocols: preincubation of PAVs with venom, and i.v. pretreatment with PAVs, prior to the venom inoculation. In this investigation, we used doses of PAVs ranging from 0.4 to 4.0mL/mg of venom equivalent up to 10 times the amount recommended by the producers for the clinical practice in Brazil. In our preincubation protocol in vivo, PAVs antagonized myotoxicity of B. jararacussu venom by 40-95%, while our pretreatment protocol antagonized myotoxic activity by 0-60%. Preincubation of antivenoms with B. jararaca venom antagonized its hemorrhagic activity by 70-95%, while pretreatment antagonized hemorrhagic activity by 10-50%. Although all PAVs demonstrated partial antagonism against both venoms, the magnitude of these effects varied greatly among the manufactures. The results suggest that the current clinical doses of these PAVs may have negligible antimyotoxic effect.

Medicinal plants with inhibitory properties against snake venoms.

Envenomations due to snake bites are commonly treated by parenteral administration of horse or sheep-derived polyclonal antivenoms aimed at the neutralization of toxins. However, despite the widespread success of this therapy, it is still important to search for different venom inhibitors, either synthetic or natural, that could complement or substitute for the action of antivenoms. Several plants have been utilized in folk medicine as antiophidian. However, only a few species have been scientifically investigated and still less had their active components isolated and characterized both structurally and functionally. This article presents a review of plants showing neutralizing properties against snake venoms which were assayed in research laboratories, correlating them with ethnopharmacological studies, as (i) the part of the plant used as antidote, (ii) its respective genus and family and (iii) inhibition of the main pharmacological, toxic and enzymatic activities of snake venoms and isolated toxins. Protective activity of many of these plants against the lethal action of snake venoms has been confirmed by biological assays. Compounds in all of them belong to chemical classes capable of interacting with macromolecular targets (enzymes or receptors). Popular culture can often help to guide scientific studies. In addition, biotechnological application of these inhibitors, as helpful alternative or supplemental treatments to serum therapy, and also as important models for synthesis of new drugs of medical interest, needs to be better oriented and scientifically explored.

Characterization of the acute pancreatitis induced by secretory phospholipases A2 in rats.

Acute pancreatitis (AP) is an inflammatory disease of the pancreas characterized by local inflammation and extrapancreatic effects such as lung injury. Secretory phospholipases A(2) (PLA(2)s) have been implicated in triggering AP, but their exact role to evoke AP is largely unknown. Therefore, we have tested the ability of sPLA(2)s to induce AP in rats, using venom sPLA(2)s with residual or high enzymatic activity (bothropstoxin-II and Naja mocambique mocambique venom PLA(2), respectively), as well as sPLA(2) devoid of catalytic activity (piratoxin-I). The injection of Naja m. mocambique venom PLA(2), bothropstoxin-II or piratoxin-I (300 microg/kg each) into the common bile duct increased significantly the pancreatic plasma extravasation and myeloperoxidase activity. The lung myeloperoxidase and serum amylase were also increased for all groups, although the Naja mocambique mocambique venom PLA(2) induced higher lung myeloperoxidase and serum amylase values, compared with piratoxin-I and/or bothropstoxin-II. Histopathology of pancreas and lungs in piratoxin-I-injected rats showed interstitial oedema in both tissues, and neutrophil infiltration with acinar cell necrosis in pancreas. In conclusion, sPLA(2)s induce AP in rats and the catalytic activity is not essential to induce the local effects in pancreas, although it appears to contribute partly to the remote lung injury.

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.

Antihemorrhagic, antinucleolytic and other antiophidian properties of the aqueous extract from Pentaclethra macroloba.

Several Brazilian plants have been utilized in folk medicine as active agents against various effects induced by snake venoms. The inhabitants of the Amazon region use, among others, the macerated bark of a plant popularly named "Pracaxi" (Pentaclethra macroloba Willd) to combat these effects. We report now the antihemorrhagic properties against snake venoms of the aqueous extract of Pentaclethra macroloba (EPema). EPema exhibited full inhibition of hemorrhagic and nucleolytic activities induced by several snake venoms. Additionally, partial inhibition of myotoxic, lethal, phospholipase and edema activities of snake venoms and its isolated PLA(2)s by EPema is reported. In vivo tests showed that EPema is able to totally inhibit a Bothrops jararacussu metalloprotease (BjussuMP-I) induced hemorrhage, suggesting interaction of the extract compounds with this high molecular weight protein. The extract did induce neither hemorrhage nor death in mice when administered alone by i.m. route. When administered separately by i.m. route, the extract did not induce death in mice at 12.5--300 mg/kg doses. Other assays demonstrated that EPema was unable to inhibit fibrinogenolytic and coagulant activities of Bothrops atrox venom. Although the mechanism of action of EPema is still unknown, the finding that no visible change was detected in the electrophoretic pattern of snake venom after incubation with the extract excludes proteolytic degradation as a potential mechanism. The search for new inhibitors of venom metalloproteases and DNAases are a relevant task. Investigation of snake venom inhibitors can provide useful tools for the elucidation of the action mechanisms of purified toxins. Furthermore, these inhibitors can be used as molecular models for development of new therapeutical agents in the treatment of ophidian accidents.

Alkylation of myotoxic phospholipases A2 in Bothrops moojeni venom: a promising approach to an enhanced antivenom production.

Bothrops moojeni crude venom (MjCV) and its two major toxins, namely myotoxin I (MjTX-I) and myotoxin II (MjTX-II) were alkylated by p-bromophenacyl bromide (BPB). After alkylation the i.p. LD(50) (mice) of MjCV and MjTX-I/II increased from 6.0 to 15.7mg/kg and from 8.0 to 45.0mg/kg, respectively. In addition, doses of 5x LD(50) of alkylated MjTX-I did not cause a single death in mice and no myonecrosis was detected for the alkylated toxins, although both proteins still induced edema. Antibodies to native and modified crude venom or myotoxins cross-reacted with 12 purified class II myotoxic phospholipases A(2) found in snake venoms of the genus Bothrops. Myotoxic PLA(2)s from class I and class III were not recognized by the above antibodies. These results suggest that the overall antigenic structure is conserved among class II myotoxic PLA(2)s, despite differences in their amino acid sequences. Anti-MjTX-I-BPB and anti-MjTX-II-BPB rabbit serum, obtained against the modified myotoxins, were apparently more efficient than those obtained against the native myotoxins. In neutralization experiments, pre-incubation of crude venom or isolated myotoxins with antibodies raised against the native or modified toxins inhibited their PLA(2) and myotoxic activities. Therefore, alkylation of His48 by BPB strongly reduces the local tissue damage induced by B. moojeni venom or isolated myotoxins while retaining antigenicity, which suggests a promising procedure for an enhanced antiophidian serum production for practical purposes.

Mn(2+) ions reduce the enzymatic and pharmacological activities of bothropstoxin-I, a myotoxic Lys49 phospholipase A(2) homologue from Bothrops jararacussu snake venom.

Bothropstoxin-I (BthTX-I), a myotoxic Lys49 phospholipase A(2) (PLA(2)) homologue isolated from Bothrops jararacussu snake venom, causes a range of biological effects, including myonecrosis, mouse paw edema, irreversible neuromuscular blockade and lysis of cell cultures. Among eight divalent cations assayed, Mn(2+) was the most effective in reducing mouse paw edema induced by BthTX-I (25 microg). Preincubating BthTX-I with Mn(2+) (1.0mM) reduced mouse paw edema by 70% and myotoxicity by 60% in mice injected i.m. with 50 microg toxin. Mn(2+) (50 microl of a 1mM solution) administered within 1min at the site of toxin injection was still but less effective in antagonising BthTX-I-induced myotoxicity. Mn(2+) (1.0mM) completely prevented BthTX-I (1.4 microM)-induced neuromuscular blockade in the mouse phrenic-nerve diaphragm preparation. Mn(2+) (0.25mM) protected about 85% of NB41A3 cells from lysis when coincubated with BthTX-I (1.0 microM) for 25h. Preincubation with 0.25mM Mn(2+) increased the sensitivity of the cells to subsequent lysis by BthTX-I in the absence of Mn(2+). BthTX-I (1 microM) caused extensive fatty acid release (from 0.8% of the total radiolabeled lipid in control cells to 56% with toxin) when incubated with the NB41A3 cell line for 25h. PLA(2) activity observed in cell cultures after addition of BthTX-I was considerably reduced by 0.25mM Mn(2+). Mn(2+) ions constitute a promising agent to assess the action mechanism and the effects of enzymatic inhibition on the pharmacological activity of Lys49 PLA(2) homologues.

Signal transduction pathways involved in the platelet aggregation induced by a D-49 phospholipase A2 isolated from Bothrops jararacussu snake venom.

Bothropstoxin-II (Bthtx-II), an Asp-49 phospholipase A(2) (D-PLA(2)) isolated from Bothrops jararacussu snake venom is able to induce platelet aggregation in a concentration-dependent manner. This effect was not due to the release of ADP from platelets since the aggregation was not suppressed by ADP scavenger systems. PMSF and PPACK were unable to inhibit Bthtx-II-induced platelet aggregation. Thus, a thrombin-like proaggregating activity of Bthtx-II can be excluded as its mechanism of action. On the other hand, indomethacin at low concentrations inhibited more markedly the ATP-release reaction than the aggregation induced by Bthtx-II, indicating that generation of cyclooxigenase products is not the most important event for the platelet aggregation reaction. It was also found that staurosporine and genistein suppressed both platelet aggregation and ATP-release reactions, but not the platelet shape-change induced by Bthtx-II. Substances that either directly activates adenylyl cyclase enzyme (forskolin and PGE(1)) or cell-permeant increasing agents (dibutyril-cAMP) inhibited in a concentration-dependent fashion, the platelet aggregation effects induced by the protein. It is concluded that Bthtx-II induces platelet aggregation and secretion through multiple signal transduction pathways.

Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects: I--gene expression profile of highly expressed phospholipases A2.

Snake venom glands are a rich source of bioactive molecules such as peptides, proteins and enzymes that show important pharmacological activity leading to in local and systemic effects as pain, edema, bleeding and muscle necrosis. Most studies on pharmacologically active peptides and proteins from snake venoms have been concerned with isolation and structure elucidation through methods of classical biochemistry. As an attempt to examine the transcripts expressed in the venom gland of Bothrops jararacussu and to unveil the toxicological and pharmacological potential of its products at the molecular level, we generated 549 expressed sequence tags (ESTs) from a directional cDNA library. Sequences obtained from single-pass sequencing of randomly selected cDNA clones could be identified by similarities searches on existing databases, resulting in 197 sequences with significant similarity to phospholipase A(2) (PLA(2)), of which 83.2% were Lys49-PLA(2) homologs (BOJU-I), 0.1% were basic Asp49-PLA(2)s (BOJU-II) and 0.6% were acidic Asp49-PLA(2)s (BOJU-III). Adjoining this very abundant class of proteins we found 88 transcripts codifying for putative sequences of metalloproteases, which after clustering and assembling resulted in three full-length sequences: BOJUMET-I, BOJUMET-II and BOJUMET-III; as well as 25 transcripts related to C-type lectin like protein including a full-length cDNA of a putative galactose binding C-type lectin and a cluster of eight serine-proteases transcripts including a full-length cDNA of a putative serine protease. Among the full-length sequenced clones we identified a nerve growth factor (Bj-NGF) with 92% identity with a human NGF (NGHUBM) and an acidic phospholipase A(2) (BthA-I-PLA(2)) displaying 85-93% identity with other snake venom toxins. Genetic distance among PLA(2)s from Bothrops species were evaluated by phylogenetic analysis. Furthermore, analysis of full-length putative Lys49-PLA(2) through molecular modeling showed conserved structural domains, allowing the characterization of those proteins as group II PLA(2)s. The constructed cDNA library provides molecular clones harboring sequences that can be used to probe directly the genetic material from gland venom of other snake species. Expression of complete cDNAs or their modified derivatives will be useful for elucidation of the structure-function relationships of these toxins and peptides of biotechnological interest.

cDNA sequence and molecular modeling of a nerve growth factor from Bothrops jararacussu venomous gland.

The complete nucleotide sequence of a nerve growth factor precursor from Bothrops jararacussu snake (Bj-NGF) was determined by DNA sequencing of a clone from cDNA library prepared from the poly(A) + RNA of the venom gland of B. jararacussu. cDNA encoding Bj-NGF precursor contained 723 bp in length, which encoded a prepro-NGF molecule with 241 amino acid residues. The mature Bj-NGF molecule was composed of 118 amino acid residues with theoretical pI and molecular weight of 8.31 and 13,537, respectively. Its amino acid sequence showed 97%, 96%, 93%, 86%, 78%, 74%, 76%, 76% and 55% sequential similarities with NGFs from Crotalus durissus terrificus, Agkistrodon halys pallas, Daboia (Vipera) russelli russelli, Bungarus multicinctus, Naja sp., mouse, human, bovine and cat, respectively. Phylogenetic analyses based on the amino acid sequences of 15 NGFs separate the Elapidae family (Naja and Bungarus) from those Crotalidae snakes (Bothrops, Crotalus and Agkistrodon). The three-dimensional structure of mature Bj-NGF was modeled based on the crystal structure of the human NGF. The model reveals that the core of NGF, formed by a pair of beta-sheets, is highly conserved and the major mutations are both at the three beta-hairpin loops and at the reverse turn.

Human neutrophil migration in vitro induced by secretory phospholipases A2: a role for cell surface glycosaminoglycans.

The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I), and type III- (Apis mellifera venom) secretory phospholipases A2 (sPLA2s) to induce human neutrophil chemotaxis, and the role of the cell surface proteoglycans, leukotriene B4 (LTB4), and platelet-activating factor (PAF), in mediating this migration. The neutrophil chemotaxis assays were performed by using a 48-well microchemotaxis chamber. Piratoxin-I, bothropstoxin-I, N. m. mocambique venom PLA2 (10-1000 microg/mL each), bothropstoxin-II (30-1000 microg/mL), porcine pancreas PLA2 (0.3-30 microg/mL), and A. mellifera venom PLA2 (30-300 microg/mL) caused concentration-dependent neutrophil chemotaxis. Heparin (10-300 U/mL) concentration-dependently inhibited the neutrophil migration induced by piratoxin-I, bothropstoxin-II, and N. m. mocambique and A. mellifera venom PLA2s (100 microg/mL each), but failed to affect the migration induced by porcine pancreas PLA2. Heparan sulfate (300 and 1000 microg/mL) inhibited neutrophil migration induced by piratoxin-I, whereas dermatan sulfate and chondroitin sulfate (30-1000 microg/mL each) had no effect. Heparitinase I and heparinase (300 mU/mL each) inhibited by 41.5 and 47%, respectively, piratoxin-I-induced chemotaxis, whereas heparitinase II and chondroitinase AC failed to affect the chemotaxis. The PAF receptor antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thienol-[3,2-f] -triazolo-[4,3-a] -diazepine-2-yl]-1-(4-morpholynil)-1-propionate) (0.1-10 microM) and the LTB4 synthesis inhibitor AA-861 [2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone] (0.1-10 microM) significantly inhibited the piratoxin-I-induced chemotaxis. Piratoxin-I (30-300 microg/mL) caused a concentration-dependent release of LTB4. Our results suggest that neutrophil migration in response to sPLA2s is independent of PLA activity, and involves an interaction of sPLA2s with cell surface heparin/heparan binding sites triggering the release of LTB4 and PAF.

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom.

An acidic (pI approximately 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an approximately 13.7kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 1SLWQFGKMINYVM-GESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0A resolution. These crystals are monoclinic and have unit cell dimensions of a=33.9, b=63.8, c=49.1A, and beta=104.0 degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 times more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation. Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies.

Chemical modifications of phospholipases A2 from snake venoms: effects on catalytic and pharmacological properties.

Phospholipases A2 (PLA2s) constitute major components of snake venoms and have been extensively investigated not only because they are very abundant in these venoms but mainly because they display a wide range of biological effects, including neurotoxic, myotoxic, cytotoxic, edema-inducing, artificial membrane disrupting, anti-coagulant, platelet aggregation inhibiting, hypotensive, bactericidal, anti-HIV, anti-tumoral, anti-malarial and anti-parasitic. Due to this functional diversity, these structurally similar proteins aroused the interest of many researchers as molecular models for study of structure-function relationships. One of the main experimental strategies used for the study of myotoxic PLA2s is the traditional chemical modification of specific amino acid residues (His, Met, Lys, Tyr, Trp and others) and examination of the consequent effects upon the enzymatic, toxic and pharmacological activities. This line of research has provided useful insights into the structural determinants of the action of these enzymes and, together with additional strategies, supports the concept of the presence of 'pharmacological sites' distinct from the catalytic site in snake venom myotoxic PLA2s.

Participation of Na(+) channels in the potentiation by Tityus serrulatus alpha-toxin TsTx-V of glucose-induced electrical activity and insulin secretion in rodent islet beta-cells.

The effects of TsTx-V, an alpha-toxin isolated from Tityus serrulatus venom, on electrical activity and insulin secretion by rodent pancreatic islet cells were studied. TsTx-V (5.6 microg/ml) depolarized mouse pancreatic beta-cells, diminished the membrane input resistance and increased the duration of the active phase of glucose-induced electrical activity. Similar results were observed with the Na(+) channel agonist veratridine (110 microM). Both agents potentiated glucose (8.3 mM)-induced insulin secretion in rat islet. In the presence of TsTx-V or veratridine, insulin secretion increased 2- and 1.4-fold over basal values, respectively (P<0.001). The Na(+) channel antagonist tetrodotoxin (6 microM) significantly decreased glucose- and TsTx-V-induced insulin secretion (P<0.001). TsTx-V also stimulated insulin secretion at low glucose concentrations (2.8 mM) whereas the beta-toxin, Ts-gamma (gamma toxin), also obtained from Tityus serrulatus venom, significantly reduced TsTx-V-induced secretion at sub- and suprathreshold concentrations of glucose. These results are consistent with a model whereby Na(+) channels participate in glucose-induced electrical activity. Alteration in the activity of these channels changes the length of time during which the beta-cell depolarizes, thereby altering the secretory behavior of the cell. The construction of a three-dimensional model for TsTx-V revealed a conserved core containing an alpha-helix and three beta-strands, with minor differences when compared with toxins from other scorpion venoms.

Bactericidal and neurotoxic activities of two myotoxic phospholipases A2 from Bothrops neuwiedi pauloensis snake venom.

Two basic myotoxic PLA(2)s, namely BnpTX-I and II, were isolated from Bothrops neuwiedi pauloensis snake venom through three chromatographic steps: ion-exchange chromatography on CM-Sepharose, gel filtration on Sephadex G-50 and reverse phase HPLC on a C18 column. Both PLA(2)s showed a M(r) around 14,000 for the monomer and 28,000 for the dimer (as estimated by SDS-PAGE), pI approximately 7.8 and approximately 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with Asp49 basic myotoxic PLA(2)s from other snake venoms. The catalytic and anticoagulant activities of BnpTX-I were higher than those of BnpTX-II. Both were able to induce cytotoxicity in vitro, as well as, myotoxicity, edema and lethality in mice. BnpTX-I also induced neurotoxic effect on mouse neuromuscular preparations and bactericidal activity on Eschericia coli and Staphylococcus aureus. After chemical modification of BnpTX-I with BPB or incubation with EDTA or Mn(2+) ions, the catalytic activity was completely abolished, while the toxic and pharmacological activities were partially reduced. Interaction with heparin inhibited the cytotoxic and bactericidal effects. Anti-BthTX-I, anti-BthTX-II and anti-115-129-C terminal antibodies strongly recognize both BnpTX-I and II. It is shown that the neurotoxic effect induced by B. neuwiedi pauloensis venom is due to the presence of myotoxic PLA(2)s. The data also corroborate the hypothesis of a partial dissociation between toxic and enzymatic domains. In addition, BnpTX-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.

Spectroscopic analysis of the stability of bothrops myotoxic phospholipases A2 to guanidine and urea denaturation.

Spectrophotometric profiles representing the unfolding induced by guanidine on Bothrops moojeni myotoxins-I (MjTX-I) and II (MjTX-II), Bothrops jararacussu bothropstoxin-I (BthTX-I) and Bothrops pirajai piratoxin-I (PrTX-I) were obtained and compared with those obtained with bovine ribonuclease A (RNAse) and trypsin. The molar (epsilon(1M)) and percent (epsilon(1%)) extinction coefficients were determined for the four myotoxins as well as for RNAse and trypsin as reference parameters. These coefficients were then used throughout this work. The changes in free energy (deltaGD(H)(2)(O)) corresponding to zero guanidine concentration and the guanidine concentrations (D(1/2)) able to convert 50% of the molecules from the native to the unfolded state were determined. The values of deltaGD (H)(2)(O) ranged from 4.42 (BthTX-I) to 8.02 (MjTX-I) kcal/mole, compared with 6.47 and 6.88 kcal/mole for trypsin and RNAse, respectively. The values for deltaGD(H)(2)(O) and D1/2 showed that BthTX-I is the least stable among the four myotoxins assayed, with a D1/2 close to that of RNAse, while MjTX-II is conformationally the most stable. Monitoring of the unfolding of RNAse and PrTX-I by a 0 to 6 M urea gradient PAGE revealed transitions from the native (N) to the unfolded (U) state with deltaG(N-U)of 0.22 and 0.41 kcal/mole, respectively. Sigmoidal curves showed well-defined two-stage transitions for both proteins.

Cloning and identification of a complete cDNA coding for a bactericidal and antitumoral acidic phospholipase A2 from Bothrops jararacussu venom.

In order to better understand the function of acidic phospholipases A2 (PLA2s) from snake venoms, expressed sequence tags (ESTs) that code for acidic PLA2s were isolated from a cDNA library prepared from the poly(A)+ RNA of venomous glands of Bothrops jararacussu. The complete nucleotide sequence (366 bp), named BOJU-III, encodes the BthA-I-PLA2 precursor, which includes a signal peptide and the mature protein with 16 and 122 amino acid residues, respectively. Multiple comparison of both the nucleotide and respective deduced amino acid sequence with EST and protein sequences from databases revealed that the full-length cDNA identified (BOJU III--AY145836) is related to an acidic PLA2 sharing similarity, within the range 55-81%, with acidic phospholipases from snake venoms. Moreover, phylogenetic analysis of amino acid sequences of acidic PLA2s from several pit viper genera showed close evolutionary relationships among acidic PLA2s from Bothrops, Crotalus, and Trimeresurus. The molecular modeling showed structural similarity with other dimeric class II PLA2s from snake venoms. The native protein BthA-I-PLA2, a nontoxic acidic PLA2 directly isolated from Bothrops jararacussu snake venom, was purified and submitted to various bioassays. BthA-I-PLA2 displayed high catalytic activity and induced Ca2+-dependent liposome disruption. Edema induced by this PLA2 was inhibited by indomethacin and dexamethasone, thus suggesting involvement of the cyclo-oxygenase pathway. BthA-I-PLA2 showed anticoagulant activity upon human plasma and inhibited phospholipid-dependent platelet aggregation induced by collagen or ADP. In addition, it displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and antitumoral effect upon breast adrenocarcinoma as well as upon human leukemia T and Erlich ascitic tumor. Following chemical modification with p-bromophenacyl bromide, total loss of the enzymatic and pharmacological activities were observed. This is the first report on the isolation and identification of a cDNA encoding a complete acidic PLA2 from Bothrops venom, exhibiting bactericidal and antitumoral effects.