Inflammation and coagulation crosstalk induced by BJcuL, a galactose-binding lectin isolated from Bothrops jararacussu snake venom.

Inflammation and coagulopathies are important systemic events following snakebite. Snake venom galactoside-binding lectins (SVgalLs) are known modulators of the immune response with no direct effect on hemostasis. Considering the crosstalk between inflammation and coagulation, the present study investigated how BJcuL, a proinflammatory SVgalL isolated from Bothrops jararacussu venom, mediated the inflammation-induced procoagulant activity. We examined the proinflammatory cytokine production and procoagulant tissue factor (TF) activity in human whole blood and monocyte-rich cell suspension (MR-PBMC) treated with BJcuL. This lectin increased production of the cytokines TNF-α, IL-1ß and IL-6, upregulated TF expression on the cell surface, and induced procoagulant activity. The proinflammatory behavior was mediated by the direct interaction between the lectin and toll-like receptor 4, via binding to ß-galactoside-containing glycoconjugates on the cell surface, and activation of NFκ-B signaling. Interestingly, the BJcuL-induced inflammation was directly associated with the procoagulant activity of MR-PBMC cells. In whole blood culture, the lectin exhibited similar behavior, i.e. it induced cytokine production and MR-PBMC TF-mediated procoagulant activity. Therefore, the present study is the first report on the inflammation-induced procoagulant activity of SVgalLs, and it indicates that BJcuL is an important factor associated with coagulopathy in patients with snake envenomation.

Cytotoxic, genotoxic, and oxidative stress-inducing effect of an l-amino acid oxidase isolated from Bothrops jararacussu venom in a co-culture model of HepG2 and HUVEC cells.

Hepatocellular carcinoma incidence rates have increased worldwide, which encouraged the development of new chemotherapeutic drugs. l-Amino acid oxidases from snake venoms are cytotoxic towards human tumor cells in in vitro monoculture systems, which do not simulate the tumor microenvironment. We examined the antitumor potential of BjussuLAAO-II, an l-amino acid oxidase from Bothrops jararacussu venom, in hepatocarcinoma cells (HepG2) in monoculture and co-culture with human umbilical vein endothelial cells (HUVEC) in vitro. All the concentrations tested (0.25-5.00 µg/mL) were cytotoxic (MTT and clonogenic survival assays) towards HepG2 and HUVEC cells in monoculture, and increased oxidative stress by 2',7'-dichlorofluorescin diacetate fluorescence assay. Only 1.00 and 5.00 µg/mL exerted these effects in HepG2 cells co-cultured with HUVEC cells, and were genotoxic (comet assay) to HUVEC cells in monoculture. BjussuLAAO-II at 5.00 µg/mL induced DNA, but not chromosomal damage (micronucleus assay) in HepG2 cells in mono- and co-culture. The cytotoxicity and genotoxicity was more pronounced in monoculture, indicating that the tumor microenvironment influences the cellular response. BjussuLAAO-II caused cell death and DNA damage in HepG2 cells in vitro by inducing oxidative stress. Therefore, BjussuLAAO-II is a promising molecule for the development of new antitumor drugs.

Antineurotoxic activity of Galactia glaucescens against Crotalus durissus terrificus venom.

Ethanolic extract of leaves of Galactia glauscescens (GGE) at concentration of 100 and 500 microg/ml prevented the neuromuscular paralysis induced by Crotalus durissus terrificus venom on mouse phrenic nerve-diaphragm preparation.

The toxin BjussuLAAO-II induces oxidative stress and DNA damage, upregulates the inflammatory cytokine genes TNF and IL6, and downregulates the apoptotic-related genes BAX, BCL2 and RELA in human Caco-2 cells.

Colorectal carcinoma is one of the most common cancers in adults. As chemotherapy, the first-choice treatment for colorectal carcinoma, is often infeasible due to acquired tumor resistance and several adverse effects, it is important to discover and explore new molecules with better therapeutic action. Snake venom toxins have shown promising results with high cytotoxicity against tumor cells, but their mechanisms of action remain unclear. Here we examined how BjussuLAAO-II, an L-amino acid oxidase isolated from Bothrops jararacussu snake venom, exerts cytotoxicity towards colorectal adenocarcinoma human cells (Caco-2) and human umbilical vein endothelial cell line (HUVEC). A 24-h treatment with BjussuLAAO-II at 0.25 - 5.00 µg/mL diminished cell viability by decreasing (i) mitochondrial activity, assessed by reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and resazurin; (ii) the activity of acid phosphatases; and (iii) lysosomal function, assessed by neutral red uptake. BjussuLAAO-II also increased intracellular levels of reactive oxygen species and DNA damage, as assessed by fluorescence and the comet assay, respectively. BjussuLAAO-II altered the expression of cell proliferation-related genes, as determined by RT-qPCR: it elevated the expression of the inflammatory cytokine genes TNF and IL6, and lowered the expression of the apoptotic-related genes BAX, BCL2, and RELA. Therefore, BjussuLAAO-II induces Caco-2 cells death by acting on multiple intracellular targets, providing important data for further studies to assess whether these effects are seen in both tumor and normal cells, with the aim of selecting this drug for possible therapeutic purposes in the future.

Isolation and characterization of TsTX-V, a new neurotoxin from Tityus serrulatus scorpion venom which delays the inactivation of Na+ channels.

TsTX-V, a new neurotoxin from Tityus serrulatus scorpion venom able to induce a prolongation of the inactivation of Na+ channels, has been purified to homogeneity. The venom was chromatographed on CM-cellulose-52 and 13 fractions were first collected. A subsequent stepwise elution chromatography of fraction XI afforded, among other toxins, highly purified TsTX-V, which showed a single band by PAGE, SDS-PAGE or isoelectric focusing, a distinctive amino acid composition, mol. wt. = 7230, pI = 8.0 and i.v. LD50 = 94 +/- 7 micrograms/kg in mice. TsTX-V induced a long lasting hypertension in anesthetized rats and prolonged the action potential of the B fibers of the rabbit vagus nerve at 0.03 microgram/ml. At 0.3 microgram/ml and higher concentrations it caused also a nerve depolarization. These effects on nerve membranes were irreversible and could be suppressed by tetrodotoxin (200-500 nM). Nerve fibers depolarized by high extracellular K+(15-30mM) concentrations still displayed long duration action potentials after TsTX-V treatment. It is suggested that TsTX-V blocks the Na+ channel inactivation system probably as an alpha-toxin.

A tripeptide isolated from Bothrops atrox venom has neuroprotective and neurotrophic effects on a cellular model of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder; however, there is no treatment able to prevent the loss of dopaminergic neurons or its consequences. Trophic factors such as NGF and BDNF has positive effects on different disorders of the brain, including neurodegeneration. Additionally, studies have suggested the use of venom peptides as a therapeutic strategy for neurological disorders. Therefore, in the present study, we investigated the neuroprotective activity of a peptide isolated from Bothrops atrox venom and its trophic ability by using a cellular model of dopaminergic neurotoxicity induced by 1-methyl-4-phenylpyridinium (MPP(+)) in PC12 cells. We showed that it decreased the activities of the apoptotic proteases caspase-9 (mitochondrial) and caspase-3 (executor) and increased cell viability and proliferation in this model. Additionally, it increased neuritogenesis in non-treated PC12 cells (neuronal model) as well as in PC12 cells treated with the dopaminergic neurotoxin. The amino acid sequence of the peptide was identified as Glutamic acid-Valine-Tryptophan (Glu-Val-Trp). These findings suggest that this tripeptide has the potential to protect against the dopaminergic neurons loss and that trophic stimulation of neuroplasticity might be involved in its mechanism of neuroprotection.

Isolation, characterization and biological activity of acidic phospholipase A2 isoforms from Bothrops jararacussu snake venom.

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIB diffracted beyond 1.8 A resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 A, b = 54.2 A and c = 90.7 A. The crystal structure has been refined to a crystallographic residual of 16.1% (R(free) = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities.

Isolation and characterization of toxic proteins from the venom of the Brazilian scorpion Tityus serrulatus.

Through gel filtration on Sephadex G-25 and chromatography on CM-cellulose-52 five toxic proteins, electrophoretically pure, were isolated from the venom of the Brazilian scorpion Tityus serrulatus and partially characterized, as follows: 1. Toxin T1 VIII, with 61 amino acid residues, mol. wt 6675 and amino terminal sequence Lys-Glx-Gly-Tyr-Leu-Met-Asx-His-Glx-Gly-Cys-Lys-; 2. Toxin T1VI with amino acid residues, mol. wt. 7549 and amino terminal sequence Gly-His-Phe-Gly-Lys; 3. Toxin T2III(I), with 63 amino acid residues, mol. wt. 7216 and amino terminal sequence Lys-Lys-Asx-Gly-Tyr-Pro-Val-Cys-Cys-Ser-; 4. Toxin T2IV, which is apparently identical to toxin T1VIII above, since it showed the same elution volume in chromatography on CM-cellulose-52, the same N-terminal Lys and the same electrophoretic mobility as T1VIII; 5. Toxin T1IV, a not previously described toxin from the venom of T. serrulatus, with 45 amino acid residues, mol. wt. 5188 and amino terminal sequence Lys-Glx-Gly-Tyr-Leu-, identical to the first five residues of T1VIII, although with a lower molecular weight. The pharmacological study of T1VIII in guinea pig vas deferens showed a pre-junctional sensitizing action, evidenced by a decrease of the dose-response curves to adrenaline and acetylcholine, with no increase of the maximum. This effect may be due to the liberation of noradrenaline.

What is tityustoxin?

Tityus serrulatus scorpion venom was fractionated by gel filtration affording two heterogeneous toxic fractions, T1 and T2; the latter was further fractionated by ion-exchange chromatography. The fraction of T2 eluted with 0.15 M ammonium acetate buffer, originally named 'tityustoxin', was shown to be a pool of several proteins. One of them, TsTX, as well as T1, was also named 'tityustoxin'. The major and perhaps most potent toxin of the venom, gamma-toxin, was eluted with 0.30 M buffer as a highly purified protein and shown to be different from TsTX. gamma-Toxin is contained in both T1 and T2.

Further characterization of toxins T1IV (TsTX-III) and T2IV from Tityus serrulatus scorpion venom.

Toxins T1IV (TsTX-III) and T2IV have been purified to homogeneity from Tityus serrulatus scorpion venom and further characterized. Their amino acid composition and SDS-PAGE reveal an approximate mol. wt of 7000. Their intracisternal LD50 (micrograms/kg) in mice were 12.9 +/- 1.6 and 3.0 +/- 0.5, while their N-terminal amino acid sequences were K-E-G-Y-A-M-D-H-E-G-C-K-F-S- and K-E-G-Y-L-M-D-H-E-G-C-K-L-S-C-F-I-R-P-S-G-Y-C-G-R-E-, respectively. This sequence of T2IV, its amino acid composition and its chromatographic and electrophoretic behaviour identify it as toxin gamma (TsTX-I), which is the major toxin from this venom. TsTX-III (13 to 102 micrograms/kg) produced a long lasting enhancement of the hypertensive effect of noradrenaline and a slight decrease of the hypotensive effect of acetylcholine, while T2IV (115 micrograms/kg) induced a prolonged hypotensive effect on the anesthetized rat. On the isolated guinea-pig vas deferens, TsTX-III (2.1 and 3.0 micrograms/ml) produced a horizontal shift of the dose-response curve for noradrenaline to the left with no change of the maximal response. At a concentration of 1.43 microM, it induced a prolongation of the duration of the B component of the compound action potential. This prolongation was strongly reduced after addition of tetrodotoxin.

A simplified procedure for the fractionation of Tityus serrulatus venom: isolation and partial characterization of TsTX-IV, a new neurotoxin.

Five toxins from the venom of the Brazilian scorpion Tityus serrulatus were purified to homogeneity by a combination of ion exchange chromatography with ammonium bicarbonate buffer (pH 7.8) on CM-cellulose-52 and rechromatography on the same resin equilibrated with ammonium acetate buffer (pH 4.7). Four of these proteins, obtained in one or two steps in high yield and lethality (named toxins IX3, IX5, and X4 and XIII) were shown to be identical with other toxins already described. A fifth one, TsTX-IV, is reported as a new toxin. Except for IX3, which showed Gly as the sole N-terminal residue, the other four toxins showed Lys. TsTX-IV has an approximate mol. wt of 6880, an i.v. LD50, in mice, of 826 +/- 156 micrograms/kg and an intracisternal LD50 of 11 +/- 9 micrograms/kg, compared to 375 +/- 45 and 4.9 +/- 0.8, respectively, for the whole venom extract. It has 61 amino acid residues and an amino acid composition different from that of any other toxin from Tityus serrulatus venom so far described. Toxins IX5, TsTX-IV and XIII induced a prejunctional type of supersensitivity on the guinea pig vas deferens, probably due to an increased release of noradrenaline.

Isolation and characterization of a new clotting factor from Bothrops jararacussu (jararacuçu) venom.

A detailed procedure for the isolation of a new clotting enzyme from the venom of Bothrops jararacussu (common name jararacuçu) is described. The estimated mol. wt of the native protein was 30,100 but 37,500 after reduction by dithiothreitol. Two major close bands corresponding to pI 5.18 and 5.20 were detected by electrofocusing but, after methanolysis, a single band focused at pI 8.20. The mol. wt of the protein moiety of this glycoprotein was 28,500, showing V-V-G-A-D-N-C-N-F-N... as N-terminal sequence. The content of neutral sugar was 4.8% and that of total sugars 5.3%. This clotting factor degraded only the A alpha-chain of the fibrinogen molecule. The stability of the clot, when produced in the presence of aprotinin opens new uses for snake clotting enzymes in the production of fibrin glue.

Biochemical and histopathological alterations induced in rats by Tityus serrulatus scorpion venom and its major neurotoxin tityustoxin-I.

Intravenous injection into the rat of sublethal doses of Tityus serrulatus scorpion venom (100 micrograms protein/kg) or its major neurotoxin tityustoxin-I (TsTX-I, 20 micrograms/kg) caused, 30-180 min after injection, statistically significant increases in the serum levels of aspartate aminotransferase, amylase, creatine kinase and lactate dehydrogenase, as well as hyperglycemia, a high level of plasma free fatty acids and a low level of liver glycogen. The in vitro serum levels of the above enzymes did not change. For alanine aminotransferase, gamma-glutamyl transferase and alkaline phosphatase, neither in vitro nor in vivo alterations were observed. The whole venom and TsTX-I caused hepatic congestion with hemolysis and hydropic degeneration. Other histological lesions included edema and congestion with subpleural hemorrhage in the lungs, hypertrophy of fibers with degeneration areas in the heart, and congestion and hemorrhage in the kidneys. In the salivary glands, alterations to the acini and ductules were visible. In the adrenal glands no morphological alterations could be detected at the studied doses. The results suggest that the in vivo enzymatic and histopathological alterations are due to tissue lesions evoked by the whole venom and TsTX-I. An indirect effect, however, induced by stimulation of acetylcholine and catecholamine release in the postganglionic nerve terminals, cannot be excluded.

A hyaluronidase from Tityus serrulatus scorpion venom: isolation, characterization and inhibition by flavonoids.

The purification procedure of a hyaluronidase from Tityus serrulatus scorpion venom is described. It involves basically an ion-exchange chromatography on CM-cellulose at pH 7.8 followed by a rechromatography of the active fraction on the same column at pH 4.7. The optima pH and temperature for maximum activity of the isolated enzyme was 6.0 and 40 degrees C, respectively. Its K(M) was 69.7 microg/ml at 37 degrees C and its specific activity was 19,900+/-1,730 turbidity reducing units (TRU)/mg against 845+/-88TRU/mg for the whole desiccated venom, representing a 23- to 24-fold purification range. The hyaluronidase activity of the purified protein (51kDa) was inhibited by some flavonoid compounds. This article also showed that T. serrulatus hyaluronidase affected on the activity of the venom's major toxin, tityustoxin-I (TsTX-I or Ts1), as reflected by alterations in the serum levels of creatine kinase (CK), lactate dehydrogenase (LD) and aspartate aminotransferase (AST) following injection of TsTX-I, in the presence or absence of hyaluronidase.

Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity.

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Pará) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C164 I165M166 motif that is associated with a "Met-turn" structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMPα-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 µg, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both α and ß-chains of the fibrinogen molecule, and it can be inhibited by EDTA, EGTA and ß-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity.

Low-molecular-mass peptides from the venom of the Amazonian viper Bothrops atrox protect against brain mitochondrial swelling in rat: potential for neuroprotection.

The neurodegenerative diseases are important causes of morbidity and mortality in Western countries. Common mechanisms of toxicity involving mitochondrial damage have been suggested; however, a definitive treatment has not yet been found. Therefore, there has been great interest in the development of mitochondria-targeted protective compounds for the treatment of neuropathies. Animal toxins represent a promising source of new molecules with neuroprotective activity and potential to originate new drugs. We present here the effects of a low-molecular-mass peptides fraction (Ba-V) from Bothrops atrox snake venom, on rat brain mitochondrial function. Ba-V did not induce the mitochondrial swelling and moreover, was as effective as cyclosporin A (CsA) to inhibit the calcium/phosphate-induced swelling, which indicates its potential to prevent the mitochondrial permeability transition (MPT). The membrane electrochemical potential, the oxygen consumption during states-3 and -4 respirations as well as the respiratory control ratio (RCR) were not affected by Ba-V. Additionally, Ba-V did not induce reactive oxygen species (ROS) generation. Interestingly, Ba-V did not protect against the generation of ROS induced by t-BOH, which suggests a protection mechanism other than ROS scavenging. Given the important role of the mitochondrial damage and, more specifically, of MPT, in the development of neuropathies, Ba-V might be useful in the future strategies for the treatment of these diseases.

Evaluation of the effect of aqueous extract of Croton urucurana Baillon (Euphorbiaceae) on the hemorrhagic activity induced by the venom of Bothrops jararaca, using new techniques to quantify hemorrhagic activity in rat skin.

Aqueous extracts of Croton urucurana (Sangra D'agua), a plant popularly considered a cicatrizant, were analyzed for anti-Bothrops jararaca venom activity. The plant extracts antagonized the hemorrhagic activity of the venom and proanthocyanidins were involved in this activity. Two new methods for the quantification of hemorrhagic activity evoked by bothropic venoms were employed. The first consists of graphic computer analysis of the hemorrhagic halo evoked in rats by dorsal intradermic administration of venom. The second method involves quantification of the hemoglobin present in the hemorrhagic halo. Based on the results, we suggest that these methods, easily implemented in the laboratory routine, allow for quantification of venom-induced hemorrhagic activity. In addition, this study demonstrates that the rich extracts of proanthocyanidins are powerful inhibitors of bothropic venom metalloproteinases.

Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae).

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA2 activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 microg/ml) plus AE (400 microg/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 microg/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 microg/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 microg/ml, 1 h) did not reveal any change in muscle fibers, but severe necrosis induced by BV or toxins (20 microg/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA2 activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 microg of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 microg), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with 1 h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors.

Tityus gamma toxin, a high affinity effector of the Na+ channel in muscle, with a selectivity for channels in the surface membrane.

Toxin gamma from the venom of Tityus serrulatus scorpion produces a partial block of the surface Na+ channel in frog muscle. This block occurs with no change in the voltage-dependence or in the kinetics of the remaining surface Na+ current. The partial blockade of Na+ channel activity occurs with no change in tubular Na+ currents nor in twitch tension. The maximum effect of the toxin is attained at concentrations as low as 3 X 10(-10) M. Hyperpolarization to potentials more negative than the resting potential (E = -90 mV) reduces or abolishes the effect of the toxin. Radioiodinated toxin gamma binds to frog muscle membranes with a very high affinity corresponding to a dissociation constant of about 1 X 10(-11) M. Data obtained with both rabbit and frog muscle indicate that toxin gamma is specific for Na+ channels in surface membranes. Toxin gamma does not seem to bind to Na+ channels in T-tubule membranes. The biochemical data are in good agreement with electrophysiological studies and data on contraction. There is one Tityus gamma toxin binding site per tetrodotoxin binding site in surface membranes. Competition experiments have confirmed that Tityus gamma toxin binds to a new toxin receptor site on the Na+ channel structure. This site is the same that the toxin II from Centruroides suffusus binding site, but this toxin has 100 times less affinity for the Na+ channel than Tityus gamma toxin.

Assignment of the disulfide bridges in bothropstoxin-I, a myonecrotic Lys49 PLA2 homolog from Bothrops jararacussu snake venom.

Bothropstoxin-I (BthTX-I), a Lys49 phospholipase A2 homolog with no apparent catalytic activity, was first isolated from Bothropsjararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29. 44. 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys5O-Cysl31, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-I disulfide bonds follow the normal pattern of group II PLA2s.