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PubMed1990

Effects of crotoxin on the isolated guinea pig heart.

The effects of crotoxin, isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus, were investigated on isolated guinea pig hearts, perfused with Locke solution, by the Langendorff method. The cardiac beats and the electrocardiogram were simultaneously registered and the creatine kinase (CK) activity of the perfusate measured. Crotoxin was infused (4.5 x 10(-8) M and 2.3 x 10(-7) M) into the heart during 90 min, and induced a remarkable decrease in the contractile force, without a significant reduction of heart rate, increased the P-R interval and displaced the S-T segment. The activity of CK only increased in the late phases of the experiments, when the force of contraction was below 25% of the control value. Arrhythmias were uncommon and no alterations of QRS duration or Q-Tc interval were observed. The reduction of the contractile force and the increase in CK activity were completely prevented by bovine serum albumin, whereas lanatoside C did not interfere with the toxin action. A bolus injection of crotoxin (11 +/- 2 nmoles) also induced a decrease of contractile force without reduction of heart rate. This decrease of force was partially prevented by indomethacin, but not by atropine. It is suggested that the reduction of contractile force evoked by crotoxin is due probably to release of free fatty acids and lysophospholipids (initial effect) and to a cellular lesion (late effect).

Albumins↗
PubMed1991

Serum kinetics of crotoxin from Crotalus durissus terrificus venom in mice: evidence for a rapid clearance.

We report on an ELISA for the detection of crotoxin with a detection limit of 1-3 pg/ml of sample. Cross-reactivity with other animal venoms occurred only at concentrations above 1 microgram/ml. Serum kinetics of crotoxin were investigated in BALB/c mice after a single 10 micrograms s.c. dose of venom obtained from Crotalus durissus terrificus. Crotoxin levels were 254 +/- 141 ng/ml serum (X +/- S.E.) 15 min after venom injection, 3.9 +/- 0.5 ng/ml serum at 30 min and undetectable thereafter. The rapid clearance of crotoxin from the serum suggests that the test may be unsuitable for the clinical management of envenomation victims.

Animals↗
PubMed1995

Binding proteins on synaptic membranes for crotoxin and taipoxin, two phospholipases A2 with neurotoxicity.

Crotoxin and taipoxin are both neurotoxic phospholipases A2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A2. Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca(2+)-free condition, taipoxin or crotoxin inhibits with IC50 of 20-1000 nM the Na(+)-dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Comparative spectroscopic studies of four crotoxin homologs and their subunits.

Structures of four related neurotoxins and their purified subunits from the venoms of Crotalus durissus terrificus, C. vegrandis, C. s. scutulatus and C. viridis concolor were examined by circular dichroism (CD), deconvolution Fourier-transform infrared (FTIR) and fluorescence spectroscopy. CD spectra suggest that in general, the isolated subunits were decreased slightly in alpha-helix, while they were increased in beta-sheet structure, relative to intact toxins. These results were consistent with FTIR results. Fluorescence quenching (50-80%) was also observed in three of the four intact toxins as compared to spectra predicted by summation of free acidic and basic subunit spectra. It was tempting to conclude from these results that major conformational changes occur in individual subunits upon formation of the dimeric toxins. Intact crotoxin, however, when exposed to urea, yields spectra (CD, FTIR and fluorescence) that are virtually identical to control intact crotoxin. These findings suggest that the enhanced fluorescence exhibited by the isolated subunits, as well as the secondary structural changes in alpha-helix and beta-sheet, are artifacts resulting from irreversible structural changes that occur during subunit isolation by urea ion-exchange chromatography. In spite of these structural changes, LD50 values of intact crotoxin reassembled from isolated subunits are unaltered from those of native crotoxin.

Animals↗
PubMed2000

Interaction of the neurotoxic and nontoxic secretory phospholipases A2 with the crotoxin inhibitor from Crotalus serum.

Crotalus durissus terrificus snakes possess a protein in their blood, named crotoxin inhibitor from Crotalus serum (CICS), which protects them against crotoxin, the main toxin of their venom. CICS neutralizes the lethal potency of crotoxin and inhibits its phospholipase A2 (PLA2) activity. The aim of the present study is to investigate the specificity of CICS towards snake venom neurotoxic PLA2s (beta-neurotoxins) and nontoxic mammalian PLA2s. This investigation shows that CICS does not affect the enzymatic activity of pancreatic and nonpancreatic PLA2s, bee venom PLA2 and Elapidae beta-neurotoxins but strongly inhibits the PLA2 activity of Viperidae beta-neurotoxins. Surface plasmon resonance and PAGE studies further demonstrated that CICS makes complexes with monomeric and multimeric Viperidae beta-neurotoxins but does not interact with nontoxic PLA2s. In the case of dimeric beta-neurotoxins from Viperidae venoms (crotoxin, Mojave toxin and CbICbII), which are made by the noncovalent association of a PLA2 with a nonenzymatic subunit, CICS does not react with the noncatalytic subunit, instead it binds tightly to the PLA2 subunit and induces the dissociation of the heterocomplex. In vitro assays performed with Torpedo synaptosomes showed a protective action of CICS against Viperidae beta-neurotoxins but not against other PLA2 neurotoxins, on primary and evoked liberation of acetylcholine. In conclusion, CICS is a specific PLA2 inhibitor of the beta-neurotoxins from the Viperidae family.

Acetylcholine↗
PubMed1979

Postsynaptic effects of crotoxin and of its isolated subunits.

Crotoxin is a potent neurotoxin from the venom of Crotalus durissus terrificus. It is composed of two subunits: a basic phospholipase A2 with low toxicity (component B) and an acidic protein seemingly devoid of intrinsic biological activity (component A). Crotoxin and its isolated phospholipase subunit block the depolarisation caused by cholinergic agonists on the isolated electroplaque from Electrophorus electricus. The other component, which is inactive when applied alone, enhances the pharmacological activity of the phospholipase when the two components are used together. Crotoxin also blocks the increase of 22Na+ efflux caused by carbamylcholine from excitable microsacs prepared from Torpedo marmorata electric organ. Crotoxin therefore acts postsynaptically, but does not interfere with the binding of alpha-toxin from Naja nigricollis to the nicotinic cholinergic receptor site. Instead, like local anesthetics, it stabilizes a desensitized form of the acetylcholine receptor characterized by its high affinity for agonists. The phospholipase component B binds in a non-saturable manner to receptor-rich membranes. In contrast, component A does not bind to acetylcholine receptor-rich membranes, but completely prevents the non-saturable binding of component B. When the two components are applied together, a saturable binding of the latter is observed with the acetylcholine receptor-rich membranes.

Animals↗
PubMed1994

The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus.

Crotoxin, the main toxin from the venom of the South American rattlesnake Crotalus durissus terrificus, is a beta-neurotoxin which consists of the non-covalent association of two subunits: a phospholipase A2 subunit B (CB), and a non-enzymic subunit A (CA). We have previously purified and characterized several isoforms of each subunit of crotoxin in the venom collected from numerous snakes. Furthermore, three cDNAs encoding two CB isoforms and the precursor, pro-CA, of subunit A have been isolated from a cDNA library prepared from a single venom gland of Crotalus durissus terrificus. The aim of this study is to analyse an individual snake venom from an animal that has been used to construct a cDNA library. Several isoforms of subunit A and two isoforms of subunit B were isolated and compared to purified and characterized subunit isoforms from pooled venom. The result of this study showed that the multiplicity and the diversity of crotoxin isoforms result from post-translational modifications occurring on a precursor and from the expression of different messenger RNAs present in an individual snake. It allowed for the identification of the two CB isoforms encoding cDNAs expressed in the individual venom with two isoforms from pooled venom, CBc and probably CBa2, that belong to two classes of crotoxin complexes which can be distinguished biochemically and pharmacologically.

Amino Acid Sequence↗
PubMed1988

Electron-microscopical study of the choroid plexus and epiplexus cells in cats following a cisternal injection of crotoxin complex.

The choroid plexus and its associated epiplexus cells in the fourth ventricle in cats were studied with scanning and transmission electron microscopy (SEM, TEM) following a cisternal injection of crotoxin complex (phospholipase A2). In SEM, the epiplexus cells of the control animals were predominantly stellate with long radiating processes. At 2 h after the administration of crotoxin complex, these radiating processes flattened out forming sheet-like membranes covering the ventricular surface of the choroid epithelial cells. The membranous coverings remained extended in 5-hour-survival cats. Numerous blebs of different sizes were observed in areas that were not covered by the cytoplasmic membrane in 5-hour animals. Some of the blebs appeared to have ruptured. In TEM, the microvilli of the choroid epithelial cells in crotoxin complex-treated rats were dilated. The luminal surface of the epithelial cells showed eruption of blebs filled with amorphous materials. Pinocytotic vesicles increased in number in the apical cytoplasm. The lumen of the ventricle often contained portions of cytoplasm believed to be derived from the extrusion of the blebs. These appeared to be engulfed by the overlying epiplexus cells. It was concluded that the injected crotoxin complex stimulated both the secretory as well as pinocytotic activity of the choroid epithelial cells. The phagocytosis of the secretory products from the epithelial cells by epiplexus cells suggests a close functional relationship between the two cell types.

Animals↗
PubMed1999

Molecular mimicry between a monoclonal antibody and one subunit of crotoxin, a heterodimeric phospholipase A2 neurotoxin.

Crotoxin is a heterodimeric phospholipase A2 neurotoxin formed by the non-covalent association of an acidic and non-toxic subunit, CA, and a basic and weakly toxic phospholipase A2, CB. The two subunits behave in a synergistic manner. CA enhances the lethal potency of CB by increasing its selectivity of action. The mAb A-56.36, directed against the non-toxic subunit CA, was previously shown to neutralize crotoxin toxicity by dissociating the crotoxin complex. In the present report, a polypeptide sequence similarity was observed between some CDRs of mAb A-56.36 and two regions of CB (pos. 60-80 and 95-110). Phage displayed peptides corresponding to VH2 and VH3 of mAb A-56.36 and to their homologous sequences in CB bind CA to different extents. This observation shows that mAb A-56.36 interacts with a region of CA involved in its interaction with CB, therefore mimicking the binding of CB to CA. A similar approach was used to determine the regions of ammodytoxin A and of agkistrodotoxin, two phospholipase A2 neurotoxins similar to CB, which are involved in the formation of heterocomplexes with CA. The analysis of these data contributes to the determination of stretches of amino acids which could constitute the paratope of mAb A-56.36, as well as the region of association of CB with CA in crotoxin.

Amino Acid Sequence↗
PubMed1992

Immunochemical detection of purified crotoxin from Crotalus durissus terrificus venom in the motor end plate of striated muscle in CBA/J mice.

Electrophysiological studies of crotoxin, a potent neurotoxic fraction from Crotalus durissus terrificus venom, have shown a pre- and post-synaptic action. In order to determine the specific site of this activity, we performed an immunohistochemical analysis on striated muscle from CBA/J mice, injected with crotoxin (5 LD50), iv, using a single-step immunoperoxidase assay with peroxidase-conjugated antibodies to whole venom. Control muscle and muscle obtained from treated animals 15 min after injection showed no staining. However, 30 min after injection, the neuromuscular motor end plate of mice who received crotoxin was clearly stained, including thin terminations, without any morphological alteration. Sixty min after administration, the motor end plate was no longer intact, but only roughly formed stained areas without clearly identifiable structures were present. These data show specific crotoxin binding to neural end plates in striated muscle, with a subsequent toxin-induced degeneration of this structure.

Animals↗
PubMed1985

Dissociation of the crotoxin complex promoted by acetylcholine.

Neuromuscular blockage at the presynaptic level is the main biological effect exerted by crotoxin, the major toxin from Crotalus durissus terrificus venom. This effect requires the dissociation of this complex at the target membrane in spite of its tightness and stability under physiological conditions of pH, ionic composition and temperature. Complex dissociation should be determined by a specific set of physicochemical conditions prevailing at the neuromuscular junction. In this regard, we have studied the effect of acetylcholine on the stability of the crotoxin complex, since this effector is present at relatively high concentrations near the presynaptic membrane of the neuromuscular junction. Evidences arising from spectrofluorometric measurements, changes in enzymatic activity and crotoxin B inactivation by b-bromophenacyl bromide indicate that acetylcholine is indeed able to promote complex dissociation at neutral pH values, apparently by titration of 2-3 carboxylate groups in crotoxin A. This effect may, at least in part, contribute in determining the target specificity of this toxin.

Acetylcholine↗
PubMed1987

A crotoxin homolog from the venom of the Uracoan rattlesnake (Crotalus vegrandis).

A major protein toxin from the venom of Crotalus vegrandis was examined by gel filtration, anion-exchange chromatography, and SDS polyacrylamide gel electrophoresis. The toxin was separated into several isoforms by ion-exchange chromatography and spontaneously dissociated into free acidic and basic subunits, mimicking the behavior of crotoxin. Rabbit antisera raised against crotoxin reacted strongly in enzyme-linked immunosorbent assays with the intact C. vegrandis toxin isoforms and their basic subunits, and formed precipitin lines of identity with intact crotoxin in double immunodiffusion gels. These results indicate that vegrandis toxin is strongly homologous with crotoxin from the venom of Crotalus durissus terrificus.

Animals↗
PubMed1992

Structure-function relationship for the highly toxic crotoxin from Crotalus durissus terrificus.

The three-dimensional structure of the highly toxic crotoxin from Crotalus durissus terrificus was modelled based on sequence analysis and the refined structure of calcium-free phospholipase of Crotalus atrox venom. Small-angle x-ray scattering experiments were performed on aqueous solutions of crotoxin. The radial distribution function derived from these scattering experiments and the one calculated from the model structure are in good agreement. Crotoxin consists of a basic and an acidic subunit. The model strongly suggests that the overall folding motif of phospholipases has been preserved in both subunits. The basic domain has an intact active site. The residues that are expected to contact the lipid tails of the phospholipid are different from other phospholipases, but they are all hydrophobic. The acidic domain consists of three independent chains interconnected by disulfide bonds. Compared to other phospholipases the active site for the greater part has been preserved in this domain, but it is not very well shielded from solvent. Most residues normally in contact with the lipid tails of the phospholipid are missing, which might explain the acidic subunit's lack of phospholipase activity. A homology between the third chain of the acidic domain and neurophysins suggests that the acidic domain may act as a chaperone for the basic domain.

Amino Acid Sequence↗
PubMed1984

Cellular and mitochondrial changes induced in the structure of murine skeletal muscle by crotoxin, a neurotoxic phospholipase A2 complex.

Following a single i.m. injection of a sublethal dose of crotoxin into the hindlimb of mice, degenerative changes were observed in the soleus muscle in the ensuing 72 hr using light and electron microscopy. A similar degree of myonecrosis resulted from the injection of the phospholipase A2 subunit alone, whereas crotoxin reconstituted from a chemically modified phospholipase A2 subunit with low catalytic activity showed weak myonecrosis and the non-enzymic subunit, crotapotin, was inactive. Visible defects of the sarcolemma were associated with underlying necrotic areas and attraction of macrophages. Internally, hypercontraction bands were often present and mitochondria in the rounded, orthodox configurational state contained linear dark bodies as part of their crystal organization. In fibre segments with slow onset of degeneration, many mitochondria additionally contained clusters of electron opaque deposit and in mitochondria, heavily laden with such deposits, energy-dispersive microprobe analysis showed the presence of high concentration of calcium. It was concluded that the primary site of action of crotoxin was hydrolysis of the sarcolemmal membrane and that mitochondrial changes were associated with calcium accumulation in response to an altered internal environment. Regeneration of fibres was rapid.

Animals↗
PubMed1990

The effects of inhibitors of arachidonate metabolism on spontaneous and evoked endplate potentials at normal and crotoxin-treated frog neuromuscular junctions.

Aspirin (1 mM), a selective cyclooxygenase inhibitor, decreased endplate potential (e.p.p.) amplitude but did not affect either the biphasic changes in m.e.p.p. frequency or triphasic changes in e.p.p. amplitude induced by crotoxin. Nordihydroguaiaretic acid (30 microM), a dual inhibitor of cyclooxygenase and lipoxygenase pathways, increased e.p.p. amplitude but did not affect the characteristic changes in m.e.p.p. frequency and e.p.p. amplitude in response to crotoxin. The incidence of giant m.e.p.p.s was reduced by aspirin and increased by NDGA. Arachidonate metabolism is not a major factor in the induction of neurotoxicity by crotoxin at the frog neuromuscular junction.

Animals↗
PubMed1990

Effects of subunit cross-linking on the properties of crotoxin.

Crotoxin was cross-linked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Cross-linked crotoxin had the expected amino-terminal amino acids, amino acid composition, behavior on SDS-PAGE and an 80% reduction of reactable lysine residues. It was also non-toxic, had reduced immunological cross-reactivity toward both poly- and monoclonal antibodies raised to the basic subunit of crotoxin and had lost greater than 95% of its phospholipase activity. Loss of toxicity is due to either subunit cross-linking or the modification of essential residues.

Amino Acids↗
PubMed1990

Isolation of a crotoxin-like protein from the venom of a South American rattlesnake (Crotalus durissus collilineatus).

1. A crotoxin-like protein was isolated from the venom of a South American rattlesnake Crotalus durissus collilineatus. 2. Many of its properties are similar to those of crotoxin, including its non-covalent heterodimeric structure, electrophoretic mobility on SDS-PAGE, isoelectric focusing properties, toxicity in mice, immunological reactivity, multiple isoforms, phospholipase activity, peptide map, and instability on an anion-exchange column. 3. Results indicate that "collilineatus toxin" is strongly homologous with crotoxin, found in the venom of Crotalus durissus terrificus, and all other characterized rattlesnake neurotoxins.

Animals↗
PubMed2001

Systemic skeletal muscle necrosis induced by crotoxin.

Systemic skeletal muscle necrosis induced by crotoxin, the major component of the venom of Crotalus durissus terrificus, was investigated. Mice received an intramuscular injection of crotoxin (0.35mg/kg body weight) into the right tibialis anterior (TA) muscles, which were evaluated 3h, 24h and 3 days later. Control mice were injected with saline. Right and left TAs, gastrocnemius, soleus and right masseter and longissimus dorsi were removed and frozen. Histological sections were stained with Toluidine Blue or incubated for acidic phosphatase reaction. Three and 24h after the injection, signals of muscle fiber injury were found: (a) in the injected TA muscles; (b) in both right and contralateral soleus and red gastrocnemius; and (c) in the masseter muscles. Contralateral TA, longissimus dorsi and white gastrocnemius muscles were not injured. In conclusion, crotoxin induced a systemic and selective muscle injury in muscles or muscle regions composed by oxidative muscle fibers.

Animals↗