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C Bon

Publications and source records attributed to C Bon.

At least 73 records · Page 4Linked to original sources
PubMed1997

Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization.

We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition (IC50 >> 10(-6) M), while Bungarus (IC50 approximately 10(-8) M) and especially Ophiophagus (IC50 < 10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody (Elec-410) previously described as a specific ligand for the Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410.

Acetylcholinesterase↗
PubMed1997

Neurotoxic phospholipases A2 ammodytoxin and crotoxin bind to distinct high-affinity protein acceptors in Torpedo marmorata electric organ.

We studied the binding of radioiodinated ammodytoxin C, a monomeric phospholipase A2 neurotoxin from Vipera ammodytes, and of radioiodinated crotoxin, a dimeric phospholipase A2 neurotoxin from Crotalus durissus terrificus, to presynaptic membranes from the electric organ of Torpedo marmorata. In both cases, two different families of specific binding sites were identified and characterized. The high-affinity binding sites for both toxins have been shown to be proteins. The low-affinity binding sites were not affected by proteinases or heat, suggesting the involvement of certain lipid structures in this type of binding. By affinity-labeling, [125I]ammodytoxin C was shown to be associated predominantly with membrane proteins of apparent molecular masses of 70,000 and 20,000 Da and to a lesser extent with several proteins of apparent molecular masses ranging between 39,000 and 57,000 Da. [125I]crotoxin, on the other hand bound primarily to a 48,000 Da membrane protein. All phospholipases A2 tested, except beta-bungarotoxin, inhibited the low-affinity specific binding of ammodytoxin C, whereas only neurotoxic phospholipases A2 prevented the high-affinity binding and the cross-linking of ammodytoxin C and crotoxin. The inhibition profiles of high-affinity binding for [125I]crotoxin and for [125I]ammodytoxin C were quite different. Ammodytoxin C and crotoxin did not inhibit each other on their respective high-affinity binding sites. These observations indicate that at least high-affinity binding sites of these two toxins are different. In contrast with crotoxin, the isolated basic subunit CB of crotoxin was able to completely inhibit the high-affinity binding of [125I]ammodytoxin C. Therefore, the acidic subunit CA of crotoxin does not simply act as a chaperone for CB subunit, but it also confers a distinct binding specificity to the crotoxin.

Affinity Labels↗
PubMed1997

Cerastatin, a new potent inhibitor of platelet aggregation from the venom of the Tunisian viper, Cerastes cerastes.

Cerastatin, a potent platelet aggregation inhibitor, was purified by gel filtration on Sephadex G-75, followed by two ion exchange chromatographies on Mono-S columns. Cerastatin is a neutral glycoprotein (pI = 6.2) of 32 kDa, made up of at least three subunits. It is devoid of phospholipase A2, esterase, fibrinogenolytic and amidolytic activities. It inhibits aggregation of washed platelets, induced by either collagen, PAF acether or thrombin, with similar IC50 of 2.3 nM. Cerastatin also inhibits the thrombin-induced clot retraction of platelet-rich plasma. It does not inhibit the amidolytic or the procoagulant activities of thrombin Cerastatin caused no lytic effect on platelet membranes since it did not cause release of lactate dehydrogenase. Pretreatment of platelets with cerastatin irreversibly inhibits the aggregation induced by thrombin. Also cerastatin completely inhibits the fibrinogen-induced aggregation of alpha chymotrypsin-treated platelets. Cerastatin therefore inhibits platelet aggregation by interfering with the interaction of fibrinogen with fibrinogen receptors.

Animals↗
PubMed1997

Procoagulant and platelet-aggregating properties of cerastocytin from Cerastes cerastes venom.

Cerastocytin is a thrombin-like serine protease with potent platelet-proaggregating properties. It is able to activate factor XIII but is less active than thrombin on plasma coagulation. The aggregation induced by cerastocytin resembles that induced by thrombin, since rabbit washed platelets desensitized by a pretreatment with thrombin do not aggregate in the presence of cerastocytin. Furthermore, preincubation of platelets with monoclonal antibodies specific for glycoproteins GPIb or GPIIbIIIa blocks receptor sites for thrombin and fibrinogen, respectively, and prevents their aggregation induced by thrombin or cerastocytin. A monoclonal antibody, inhibitor of von Willebrand factor (VWF)-dependent agglutination, blocks the aggregation induced by cerastocytin. After activation with cerastocytin, washed rabbit platelets degranulate and secrete ATP and phospholipase A2. However, cerastocytin is less potent in inducing the release of phospholipase A2 than in inducing ATP secretion.

Adenosine Triphosphate↗
PubMed1997

Distinct bothrojaracin isoforms produced by individual jararaca (Bothrops jararaca) snakes.

Bothrojaracin (apparent mol. wt 27,000) is a potent inhibitor of thrombin previously isolated from the venom of Bothrops jararaca. Several molecular variants (isoforms) have been identified in a pool of venom collected from a large number of animals. In order to determine whether an individual snake produces a single type of bothrojaracin or multiple isoforms, we analyzed the bothrojaracin content of venoms collected individually from six adult B. jararaca snakes. Bothrojaracin was found in all venoms, but its activity was especially high in three of them. After purification on an alpha-thrombin affinity column, followed by gel filtration on Superose 12 HR, proteins from these three venoms migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as single bands of 27,000 and their amino-terminal sequences (residues 1-28) revealed extensive homology with bothrojaracin. In contrast, the material purified from the three venoms with low bothrojaracin activity consisted of bothrojaracin together with inactive proteins. Differences in the sequences obtained for bothrojaracin isolated from individual venoms indicated the existence of more than one isoform in the venom of an individual snake.

Amino Acid Sequence↗
PubMed1997

Convulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets.

Convulxin, a very potent aggregating protein from rattlesnake venom, was purified by a new procedure and its heterodimeric structure alpha 3 beta 3 was confirmed. The polypeptide N-terminal sequences of convulxin subunits were determined by Edman degradation. They are very similar and appear homologous to botrocetin from Bothrops jararaca venom and to rattlesnake lectin from Crotalus atrox venom, both being classified among the C-type lectin family. The binding of 125I-labelled convulxin to blood platelets has also been analysed under equilibrium conditions. These studies indicated that convulxin binds to platelets with a high affinity (Kd = 30 pM) on a small number of binding sites (1000 binding sites per cell). The high-affinity binding of convulxin appears specific to platelets, since it is not observed on other cell types such as neutrophils and erythrocytes. Also, the high-affinity binding of convulxin to membranes platelet is not inhibited by alpha-thrombin, fibrinogen, collagen, laminin binding inhibitor, RGDS peptide, adenosine diphosphate, platelet-activating factor-acether, serotonin or epinephrine. This, together with the recent observation that platelet activation by convulxin is partially mediated by phospholipase C and involves other mechanisms as well, indicates that convulxin may interact with a specific platelet acceptor (receptor) protein which has yet to be characterized.

Amino Acid Sequence↗
PubMed1997

Structure-activity relationships in platelet activating factor. 9. From PAF-antagonism to PLA2 inhibition.

Many important mediators of inflammation result from the liberation of free arachidonic acid from phospholipid pools, which arise from the action of phospholipase A2 (PLA2). Therefore the inhibition of this enzyme would be an important treatment in many inflammatory disease states. Starting from a series of compounds which are known as PAF-antagonists, we have synthesized new molecules. These new compounds inhibited various secretory PLA2s, with IC50's in the mumol range. This allowed us to analyze the structure-activity relationships for PLA2 inhibition. The results showed that inhibition of secretory PLA2 depends on the length of the alkyl chain, with an optimum for 13 to 17 carbons, which is in agreement with X-ray crystallographic and nuclear magnetic resonance (NMR) studies on the active site of PLA2s, and that a free nitrogen on the piperazine ring is required to ensure a good inhibitory potency.

Animals↗
PubMed1997

A new phospholipase A2 inhibitor, unrelated to substrate analogues: kinetic characterization of the inhibition of secretory phospholipases A2 by PMS 832.

Starting from a series of compounds which were known to be PAF antagonists, we have synthesized molecules that are good inhibitors of PLA2s of groups I or II, with IC50 in the micromolar range (Binisti et al., 1997). In this report we investigate the mechanism of inhibition of bovine and porcine pancreatic phospholipases A2 (group I), and platelet lysate phospholipase A2 (group II) by one of these compounds, 1-(4'-methoxybenzoyl)-2-n-tridecylpiperazine (PMS 832). We show that PMS 832 behaves as a reversible, competitive inhibitor, with Ki values of 4.1 +/- 1.2 and 1.5 +/- 0.4 microM for porcine pancreatic phospholipase A2 and platelet lysate phospholipase A2, respectively. PMS 832 failed to inhibit platelet activation induced by several agonists and was also found to be inactive towards phospholipase C from Bacillus cereus, indicating a high specificity for phospholipase A2 inactivation. Thus, PMS 832 and its derivatives could serve as interesting tools to investigate the role of extracellular phospholipases A2 in inflammatory processes, and may be useful in the development of new anti-inflammatory agents.

Animals↗
PubMed1997

Biologically active human anti-crotoxin scFv isolated from a semi-synthetic phage library.

BACKGROUND: The display of repertoires of antibody fragments on the surface of filamentous bacteriophages offers a new way of making antibodies with predefined binding specificities. OBJECTIVES: Here we explored the use of this technology to find human antibodies with biological properties. Phage-scFv specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus, were isolated from a 'single pot' repertoire of more than 10(8) clones made in vitro from human V gene segments [1]. The crotoxin molecule is composed of two noncovalently linked subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component B (CB) and an acidic, nonenzymatic and nontoxic subunit called component A (CA). CA is able to increase the toxicity as well as the specificity of action of CB simultaneously reducing its enzymatic activity. STUDY DESIGN: Two clones were isolated (4-21 and 5-3-1) which are specific of the basic subunit CB, but of a moderate affinity (about 10(-7) M). Clones 4-21 and 5-3-1 have different amino acid sequences and different effects on CB properties suggesting that they are raised against different CB epitopes. Purely cholinergic synaptosomes isolated from Torpedo electric organs provide a suitable model to study the presynaptic effects of crotoxin. In this model, CB was shown to induce a larger acetylcholine release than crotoxin. RESULTS: A dose-dependent increase of acetylcholine release was observed when crotoxin was incubated with increasing amounts of phage-scFv 4-21. This clone was also shown to increase the enzymatic activity of crotoxin. These observations suggest that phage-scFv might dissociate the complex CA-CB. It could be therefore a neutralizing antibody since CB is much less toxic than crotoxin. This shows that 'single pot' libraries are capable of providing not only immunochemical reagents of high specificity but also biological reagents of high quality. The use of this library appears to open new possibilities for immune passive therapy.

Amino Acid Sequence↗
PubMed1997

Effect of antivenom on venom pharmacokinetics in experimentally envenomed rabbits: toward an optimization of antivenom therapy.

Antivenomous immunotherapy is still used empirically. To improve the efficacy and safety of immunotherapy, we studied the effects of administering antivenom antibodies (F(ab')2) on the pharmacokinetics of the Vipera aspis venom in rabbits. Free venom levels were measured by enzyme-linked immunosorbent assay and total concentrations were quantified by measuring the radioactivity of trichloroacetic acid-precipitable radioiodinated venom. The intravenous infusion of 125 mg of antivenom 7 h after intramuscular injection with 700 microg x kg(-1) of V. aspis venom produced a redistribution of the venom antigens from the extravascular to the vascular space. Moreover, anti-venom antibodies were able to neutralize the totality of venom antigens in the vascular space, because no free plasma venom was detectable by enzyme-linked immunosorbent assay within 15 min after antivenom injection. Similar effects were obtained after injection of 25 mg of antivenom; however, the venom was only partially neutralized with lower doses (5 and 2.5 mg). We further established that intravenous injection is the most efficient route for antivenom administration, and we examined the effects of early and late immunotherapy. Finally, the efficacy of Fab antibodies was compared with that of F(ab')2; the plasma redistribution and the immunoneutralization of the venom were lower than those induced after injection of the same dose of F(ab')2. The difference between the effects of F(ab')2 and Fab could be explained by the differential pharmacokinetics of the two fragments.

Animals↗
PubMed1997

[Acetylcholinesterase from snake venoms].

Acetylcholinesterase (AChE) plays a key role in cholinergic transmission. For example, located at the neuro-muscular junction of vertebrates, it allows a fine temporal control of muscle contraction. The presence of AChE in tissues devoid of cholinergic function is also well known and raises the question of its role. In particular, AChE occurs at high level in the venoms of Elapid snakes, except Mambas. In contrast, the venom of snakes belonging to Viperid or Colubrid families does not contain any AChE. AChE purified from snake venom consists of soluble, hydrophilic monomers. Cloning the cDNA of the venom AChE from Bungarus fasciatus showed that its C-terminal peptide is very different from those of other AChEs. This peptide is encoded by a new alternative exon, called S for Soluble and Snake. It is a short very basic peptide of 15 residues. Analysis of the venom enzyme and in vitro expression experiments showed that the last eight residues are removed in the mature protein. This cleavage is not necessary for enzymatic activity and occurs before secretion of the enzyme. AChEs from snake venoms vary in their sensitivity to peripheral site inhibitors, notably to Mambas toxins, fasciculins. While Ophiophagus AChE is as sensitive as Torpedo enzyme (IC50 around 10(-10) M), Naja and Heamacatus AChEs are insensitive to the toxin up to a concentration of 10(-6) M. Bungarus AChE has an intermediary IC50 of 10(-8) M. The analysis of its sequence shows two major differences, in the peripheral site region, when compared to Torpedo or mammalian AChEs: at position 70 it contains a methionine instead of a tyrosine and at position 285, it contains a lysine instead of an acidic residue (glutamic or aspartic acid). The modification of these residues by site-directed mutagenesis and the enzymatic analysis of modified recombinant enzymes confirmed the implication of these two residues in the properties of Bungarus AChE peripheral site. The presence of an alternative exon, used for generating a soluble form of AChE in venoms, raises interesting evolutionary questions: does it exist in snakes whose venom does not contain AChE, e.g. Mambas? did this exon preexist, for expression in other contexts? In addition snake venoms offer an exceptional system for analysing the mechanism of peripheral site inhibition, because of its wide range of sensitivity.

Acetylcholinesterase↗
PubMed1996

Bothrojaracin: a potent two-site-directed thrombin inhibitor.

The thrombin inhibitor, bothrojaracin [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C., & Bon, C. (1993) Biochemistry 32, 10794-10802], is a 27 kDa protein isolated from the venom of Bothrops jararaca that blocks several thrombin functions, including fibrinogen clotting, platelet activation, and fibrin and thrombomodulin binding, but does not interact with the catalytic site. In the present report, we show that the high affinity binding of alpha-thrombin to immobilized bothrojaracin (Kd = 0.6 nM) is inhibited by the C-terminal peptide of hirudin and that the gamma-cleavage within exosite 1 reduces the affinity of bothrojaracin for thrombin (Kd = 0.3 microM), indicating that bothrojaracin binding to exosite 1 is a major determinant of the thrombin-bothrojaracin interaction. In addition, we show that bothrojaracin decreases the rate of inhibition of alpha- and gamma-thrombin by the antithrombin III-heparin complex. Competition of bothrojaracin with heparin or prothrombin fragment 2 for binding to thrombin indicates that bothrojaracin not only binds exosite 1 but also binds exosite 2 or in close proximity. Bothrojaracin binds to the thrombin precursor, prothrombin. This interaction is calcium-independent and is prevented by heparin, suggesting that it is mediated by exosite 2. Bothrojaracin inhibits platelet activation induced by clot-bound thrombin and slowly dissociates thrombin from the fibrin clots. Altogether, our results indicate that the high affinity of bothrojaracin for thrombin is supported by a double-site interaction and results in an efficient inhibition of both soluble and clot-bound thrombin.

Animals↗
PubMed1996

Cloning and expression of acetylcholinesterase from Bungarus fasciatus venom. A new type of cooh-terminal domain; involvement of a positively charged residue in the peripheral site.

As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs. It is, however, associated with a short hydrophilic carboxyl-terminal region, containing no cysteine, that bears no resemblance to the alternative COOH-terminal peptides of the GPI-anchored molecules (H) or of other homomeric or heteromeric tailed molecules (T). Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion. The COS cells produced Bungarus AChE about 30 times more efficiently than an equivalent secreted monomeric rat AChE. The recombinant Bungarus AChE, like the natural venom enzyme, showed a distinctive ladder pattern in nondenaturing electrophoresis, probably reflecting a variation in the number of sialic acids. By mutagenesis, we showed that two differences (methionine instead of tyrosine at position 70; lysine instead of aspartate or glutamate at position 285) explain the low sensitivity of Bungarus AChE to peripheral site inhibitors, compared to the Torpedo or mammalian AChEs. These results illustrate the importance of both the aromatic and the charged residues, and the fact that peripheral site ligands (propidium, gallamine, D-tubocurarine, and fasciculin 2) interact with diverse subsets of residues.

Acetylcholinesterase↗
PubMed1996

Acetylcholinesterase from Bungarus venom: a monomeric species.

The venom of Bungarus fasciatus, an Elapidae snake, contains a high level of AChE activity. Partial peptide sequences show that it is closely homologous to other AChEs. Bungarus venom AChE is a non-amphiphilic monomeric species, a molecular form of AChE which has not been previously found in significant levels in other tissues. The composition of carbohydrates suggests the presence of N-glycans of the 'complex' and 'hybrid' types. Ion exchange chromatography, isoelectric focusing and electrophoresis in non-denaturing and denaturing conditions reveal a complex microheterogeneity of this enzyme, which is partly related to its glycosylation.

Acetylcholinesterase↗
PubMed1996

The anticoagulant effect of the human secretory phospholipase A2 on blood plasma and on a cell-free system is due to a phospholipid-independent mechanism of action involving the inhibition of factor Va.

Blood platelets play a central role in haemostasis by leading to plug formation and by increasing the efficiency of blood coagulation. We have previously shown that blood platelets contain a group II secretory phospholipase A2 (sPLA2 grII) which is released into the extracellular medium upon activation but is unable to stimulate blood platelets. We presently reported an investigation of the putative involvement of the human sPLA2 grII (hsPLA2 grII) in the coagulation process, both in the absence and in the presence of activated platelets. We show that this enzyme prolongs the recalcification time of blood plasma even in the presence of activated platelets. The positive action of blood platelets on coagulation is correlated, at least in part, with the appearance at the cellular surface of procoagulant phospholipids which constitute a potential target for hsPLA2 grII. We therefore investigated the involvement of its enzymatic activity in the anticoagulant effect of this enzyme. We observed that the replacement of CaCl2 by SrCl2 to initiate the coagulation cascade did not suppress, but rather increased, the inhibitory action of hsPLA2 grII. Moreover, hsPLA2 grII hydrolyzed only a minor proportion of platelet phospholipids, and it did not affect plasma phospholipids. Taken together, these observations strongly suggest that the major action of hsPLA2 grII on blood coagulation does not involve the hydrolysis of phospholipids, in contrast with the strong anticoagulant effect of the group II venom phospholipase A2 from Crotalus durrissus terrificus. We next studied which step of the coagulation cascade was affected by hsPLA2 grII. Using purified coagulation factors, we demonstrated that hsPLA2 grII strongly inhibited the prothrombinase activity. This inhibitory effect was independent of the presence of phospholipids but required factor Va, leading to the hypothesis that hsPLA2 grII inhibited this factor. Further, the anticoagulant effect of hsPLA2 grII was observed on normal and factor-X-deficient plasma, but not on factor-V-deficient plasma. In conclusion, the anticoagulant action of hsPLA2 grII is based on a nonenzymatic mechanism of action involving the inhibition of factor Va.

Animals↗
PubMed1996

4-Alkoxybenzamidines as new potent phospholipase A2 inhibitors.

A series of 4-alkoxybenzamidines was synthesized, varying the number of carbons of the alkyl chain, and their potency as phospholipase A2 (PLA2) inhibitors was evaluated. The relationship between their capacity to inhibit PLA2 activity and their lipophilicity was examined. The optimum of the inhibitory effect against two extracellular PLA2S from rabbit platelets and bovine pancreas was observed with compounds bearing an alkyl chain of 12 and 14 carbons. These 4-dodecyl and tetradecyloxbenzamidines inhibited bovine pancreatic and rabbit platelet lysate PLA2S with IC50 values of 3 microM and 5-5.8 microM, respectively. The mechanism of inhibition was of the competitive type. In addition, 4-tetradecyloxbenzamidine was shown to exert an antiinflammatory effect in vivo on the carrageenan-induced rat paw oedema. These results show that 4-tetradecyloxybenzamidine will serve as an interesting tool to investigate the physiological role of mammalian-secreted PLA2, both in vitro and in vivo.

Acute Disease↗