Green Mamba Research Articles

A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps): H. Schweitz, P. Vigne, D. Moinier, C. Frelin and M. Lazdunski (Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis 06560 Valbonne, France)

A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps): H. Schweitz, P. Vigne, D. Moinier, C. Frelin and M. Lazdunski (Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis 06560 Valbonne, France)

Actions of fasciculin-1 and -2, anticholinesterase peptides isolated from green mamba venom, on end-plate currents at the neuromuscular junction: J. Molgo, 1L. G. Magazanik, 1J. M. Hermel, 1P. Juzans, 1J. Stinnakre and E. Karlsson2 ( 1Laboratoire de Neurobiologie Cellulaire et Moléculaire, C.N.R.S., 91198-Gif sur Yvette, France; and 2Department of Biochemistry, Biomedical Center, University of Uppsala, S-75123 Uppsala, Sweden)

Actions of fasciculin-1 and -2, anticholinesterase peptides isolated from green mamba venom, on end-plate currents at the neuromuscular junction: J. Molgo, 1L. G. Magazanik, 1J. M. Hermel, 1P. Juzans, 1J. Stinnakre and E. Karlsson2 ( 1Laboratoire de Neurobiologie Cellulaire et Moléculaire, C.N.R.S., 91198-Gif sur Yvette, France; and 2Department of Biochemistry, Biomedical Center, University of Uppsala, S-75123 Uppsala, Sweden)

M1-toxin

The venom of the Eastern green mamba from Africa, Dendroaspis angusticeps, contains a number of toxins which block the binding of 3H-antagonists to genetically-defined m1 and m4 muscarinic acetylcholine receptors. Most of the anti-muscarinic activity of the venom is due to the presence of a newly-isolated toxin, “m1-toxin”, which has 64 amino acids and a molecular mass of 7361 Daltons. At present m1-toxin is the only ligand which is known to be capable of fully blocking m1 receptors without affecting m2-m5 receptors. It binds very rapidly, specifically and pseudoirreversibly to the extracellular face of m1 receptors on cells, in membranes or in solution, whether or not the primary receptor site is occupied by an antagonist. Bound toxin can either prevent the binding and action of agonists or antagonists, or prevent the dissociation of antagonists. The toxin is useful for identifying m1 receptors during anatomical and functional studies, for recognizing and stabilizing receptor complexes, and for occluding m1 receptors so that other receptors are more readily studied.

Single voltage-dependent potassium channels in rat peripheral nerve membrane.

1. Voltage-dependent potassium channels were investigated in rat axonal membrane by means of the patch-clamp recording technique. Three different types of channels (F, I and S) have been characterized on the basis of their single-channel conductance, activation, deactivation and inactivation properties. 2. The fast (F) channels were activated smoothly at potentials (E) between -50 and 50 mV (E50 = 4.6 mV). They had a conductance of 55 pS for inward current and 30 pS for outward current in solutions containing 155 mM K+ (high K+) on both sides of the membrane at 21-23 degrees C. The F-channels demonstrated the fastest deactivation, within 1-2 ms, and inactivated in a few hundreds of milliseconds. The time constant of inactivation was 143 ms at E = +40 mV. 3. The intermediate (I) channels activated steeply between E = -70 and -50 mV (E50 = -64.2 mv) and had a single-channel conductance of 33 pS for inward and 18 ps for outward currents. The I-channels deactivated with intermediate kinetics with the time constants of 20.4 ms and 10.1 ms at E = -80 mV and E = -100 mV, respectively. Complete inactivation of the channels developed over tens of seconds. The time constant of inactivation was 7.4 s at E = +40 mV. 4. The slow (S) channels were active at potentials positive to -90 mV. Their conductance was 10 pS for inward currents. The time constant of activation of the S-channels was strongly potential dependent. At a holding potential of -100 mV the channels deactivated during a long time interval between 30 ms and 1 s, producing long-lasting tail currents. The mean time constant of deactivation for S-channels was 129 ms. 5. The conductances of F- and I-channels measured under normal physiological conditions (Ringer solution in bath) were 17 and 10 pS, respectively. 6. Tetraethylammonium (TEA), the classic blocker of potassium channels, suppressed F-, I- and S-channels. It gradually reduced the apparent amplitude of unitary currents in a dose-dependent manner with IC50 equal to 1.2 mM for F-channels, 0.6 mM for I-channels and 1.4 mM for S-channels. Dendrotoxin (DTX), a toxin from the green mamba snake, considerably inhibited the I tail currents at nanomolar concentrations (IC50 = 2.8 nM) while the amplitudes of single I-channel currents were not affected. 7. The K+ channels of F, I and S types form the basis of the potassium conductivity in mammalian peripheral myelinated axon.(ABSTRACT TRUNCATED AT 400 WORDS)

1.9-A resolution structure of fasciculin 1, an anti-acetylcholinesterase toxin from green mamba snake venom.

1.9-A resolution structure of fasciculin 1, an anti-acetylcholinesterase toxin from green mamba snake venom.

A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps).

This paper describes the purification, sequence, and biological properties of a 38-amino acid residue peptide from the venom of Dendroaspis angusticeps which shared important sequence homologies with natriuretic peptides. Dendroaspis natriuretic peptide (DNP) relaxed aortic strips that had been contracted by 40 mM KCl with a potency (K0.5 = 20 nM) similar to that of atrial natriuretic peptide (ANP) and larger than that of C type natriuretic peptide (CNP). The relaxing actions of ANP and DNP (both at 100 nM) were mutually exclusive. Bovine aortic endothelial cells responded to ANP (K0.5 = 3 nM) and DNP (K0.5 = 3 nM) but not to CNP by a large activation of guanylate cyclase. Rat aortic myocytes showed larger cGMP responses to CNP (K0.5 = 10 nM) than to ANP or DNP (K0.5 = 100 nM). Finally, DNP completely prevented the specific 125I-ANP binding to clearance receptors in cultured aortic myocytes with a potency (Kd = 10 nM) that was less than that of ANP (Kd = 0.3 nM). It is concluded that DNP is a new member of the family of natriuretic peptides and that it recognizes ANPA receptors and clearance, ANPc receptors, but not CNP-specific ANPB receptors.

Crystal structure of α-dendrotoxin from the green mamba venom and its comparison with the structure of bovine pancreatic trypsin inhibitor

The three-dimensional structure of α-dendrotoxin (α-DTX) from the green mamba ( Dendroaspis angusticeps) venom has been determined crystallographically using the method of isomorphous replacement and refined at 2.2 Å resolution using a restrained least-squares method. The crystallographic R-factor is 0.169 for all 3451 measured reflections between 7.0 and 2.2 Å. Although the main-chain fold of α-DTX is similar to that of homologous bovine pancreatic trypsin inhibitor (BPTI), there are significant differences involving segments of the polypeptide chain close to the “antiprotease site” of BPTI. Comparison of the structure of α-DTX with the existing models of BPTI and its complexes with trypsin and kallikrein reveals structural differences that explain the inability of α-DTX to inhibit trypsin and chymotrypsin.

From venom to toxin to drug?

Synopsis:Venoms are complex mixtures of many different components, whereas a toxin is a single pure compound whose activity can be specified. Toxins often have novel, highly specific activities. Studies on toxins open the way to design therapeutically useful molecules based on the structural information obtained from the toxins.An example of successful drug development from a component of a snake venom is that of the inhibitors of angiotensin-converting enzyme (ACE). The first ACE inhibitors were developed from work on peptides isolated from the Brazilian arrowhead viperBothrops jaracusathat prolonged the action of bradykinin. The venom components blocked the enzyme that inactivated bradykinin; the same enzymeactivatedthe precursor of the vasoconstrictor hormone angiotensin. Hence, a valuable new type of therapy was established by developing the leads provided by the natural toxin.Dendrotoxins are small proteins from mamba venoms that block rapidly activating K+channels in neurones. There are possibilities that dendrotoxin could be the basis for drug design. A small analogue that mimicked the activity of dendrotoxin in the central nervous system might boost the activity of certain neuronal pathways. Potentially, such a compound might be able to restore some of the function of systems damaged by progressive neurodegenerative diseases. Although dendrotoxin-like compounds would not be a cure for these diseases, they may provide temporary relief from symptoms.Conversely, compounds with the specificity of dendrotoxin but with ability to activate K+channels, rather than block them, may be a novel way of reducing abnormal electrical activity in the brain. Hence, they may form the basis of a new type of anticonvulsant drug for epilepsies.Recent work at Strathclyde has concentrated on defining the structure-activity relationships of the naturally occurring dendrotoxins and on synthesising small analogues of the postulated ‘active site’. Preliminary results are encouraging, and it is hoped to go from green mamba venom to dendrotoxin to therapeutically useful drugs.

Comparison of the effects of four dendrotoxin peptides, 4-aminopyridine and tetraethylammonium on the electrically evoked [ 3H]noradrenaline release from rat hippocampus

We have examined the effects of four dendrotoxin (DaTX) peptides, α-, β-, γ- and δ-DaTX, separated from the venom of the green mamba ( Dendroaspis angusticeps), on field stimulation-evoked [ 3H]noradrenaline (NA) release from rat hippocampus and compared their effects with those of two other inhibitors of K + channels, 4-aminopyridine (4-AP) and tetraethylammonium (TEA). 4-AP (10–300 μM) and TEA (0.1–5 mM) facilitated the evoked [ 3H]NA release in a concentration-dependent manner. The evoked [ 3H]NA release was reduced to about half by α 2-adrenoceptor stimulation (UK 14,304; 100 nM) and this reduction was antagonized by 4-AP (10–100 μM), whereas TEA even at 5 mM was a poor inhibitor of α 2-effects. α-DaTX (10–200 nM) mimicked 4-AP in increasing the electrically evoked [ 3H]NA release and diminishing the inhibitory effects of UK 14,304 in a concentration-dependent manner. δ-DaTX did not itself alter the electrically evoked [ 3H]NA release, but at 200 nM, it reduced the effects of α 2-receptor stimulation. β- and γ-DaTX (up to 200 nM) had no significant effects. 4-AP, 3,4-diaminopyridine (3,4-DAP), TEA and the four dendrotoxins displaced the binding of [ 3H]p-aminoclonidine ([ 3H]PAC) from α 2-receptors. The IC 50 values were 6.6 × 10 −4, 1.42 × 10 −3, 5.6 × 10 −2 for 4-AP, 3,4-DAP and TEA, respectively, and 3.19 × 10 −6 M for α-DaTX. Thus, their potency as inhibitors of α 2-receptors is apparently too low to account alone for the antagonism by K + channel inhibitors of α 2-effects on NA release. In conclusion, K + channels that can be inhibited by both 4-AP and α-DaTX appear to be important in the regulation of [ 3H]NA release evoked by electrical field stimulation. The antagonism of presynaptic α 2-adrenoceptor agonists by K + channel inhibitors can be caused by a combination of direct receptor actions, increased amounts of endogenous agonist in the biophase and functional antagonism.

Two types of fast K+ channels in rat myelinated nerve fibres and their sensitivity to dendrotoxin

The effect of dendrotoxin (DTX), a component of the venom of the Eastern green mamba snake, Dendroaspis angusticeps, on K+ currents in rat myelinated nerve fibres was studied in voltage clamp experiments, immunocytochemistry and binding experiments. The analysis of K+ tail currents in 160 mM KCl solution revealed that K+ channels with slow gating kinetics predominate in the intact node of Ranvier. These slow K+ channels were not blocked by DTX. Intact nerve fibres additionally showed fast K+ tail currents of small amplitude which could be blocked by DTX. After enzymatic demyelination with pronase, fast K+ currents of large amplitude appeared. Analysis of the non-monotonous voltage dependence of the fast K+ conductance and the partial pharmacological block by DTX suggest the presence of two subtypes of fast K+ channels in rat nerve fibres similar to the Kf1 and Kf2 channels previously described in the frog and toad node of Ranvier. The DTX concentration required for 50% inhibition (IC50) for the Kf1 component was 8 nM. The IC50 of the blocked Kf2 component was the same as that for Kf1, but the Kf2 component was only partially blocked (about 50%). In contrast to frog nerve, these two fast K+ channel subtypes are located predominantly in the paranodal region. Immunocytochemical staining experiments with DTX using the peroxidase-antiperoxidase technique confirmed the electrophysiological data. In intact nodes, either no staining or only slight staining in some fibres was found. After demyelination, extensive staining of paranodal and internodal regions occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence of a snake venom toxin that binds to the muscarinic acetylcholine receptor

E. Karlsson, C. Risinger, M. Jolkkonen, C. Wernstedt and A. Adem. Amino acid sequence of a snake venom toxin that binds to the muscarinic acetylcholine receptor. Toxicon 29, 521–526, 1991.—The green mamba, Dendroaspis angusticeps, has two protein toxins that bind to the muscarinic acetylcholine receptor. The sequence of muscarinic toxin 2 was determined with an automatic gas phase sequencer. The C-terminal residue is Asp as determined by hydrazinolysis and amino acid analysis. Toxin 2 has 65 amino acid residues and a formula weight of 7040. It is homologous to a large number of other snake venom toxins as short α-neurotoxins, cardiotoxins/cytotoxins and angusticeps-type toxins of mamba venoms. The sequence is confirmed in the accompanying article ( Ducancel, F., Rowan, E. G., Cassart, E., Harvey, A. L., Menez, A. and Boulain, J.-C. Toxicon 29, 516–520, 1991).

Amino acid sequence of a muscarinic toxin deduced from the cDNA nucleotide sequence

F. Ducancel, E. G. Rowan, E. Cassar, A. L. Harvey, A. Ménez and J.-C. Boulain. Amino acid sequence of a muscarinic toxin deduced from the cDNA nucleotide sequence. Toxicon 29, 516–520, 1991.—We prepared a cDNA library from venom glands of the green mamba Dendroaspis angusticeps. A cDNA clone was isolated using an appropriate nucleotide probe. The nucleotide sequence codes for a 21 residue signal peptide followed by a 65 residue protein having the amino acid sequence of muscarinic toxin 2, as confirmed in the accompanying paper ( Karlsson, E., Risinger, C., Jolkkonen, M., Wernstedt, C. and Adem, A.). The cDNA encoding the muscarinic toxin has been compared with those encoding other snake toxins. There are close similarities with short-chain curaremimetic neurotoxins.

Preliminary X-ray analysis of crystals of fasciculin 1, a potent acetylcholinesterase inhibitor from green mamba venom

Fasciculin 1 from Dendroaspis angusticeps has been crystallized by vapour diffusion, in sodium acetate using sodium thiocyanate as precipitant. Tetragonal crystals (space group P4(1)2(1)2 or P4(3)2(1)2) diffract to 1.8 A resolution. The unit cell parameters are a = 40.4 A and c = 81.1 A. We estimated the presence of one molecule in the asymmetric unit.

A K+ channel in Xenopus nerve fibres selectively blocked by bee and snake toxins: binding and voltage‐clamp experiments.

1. The effects of mast cell degranulating peptide (MCDP), a toxin from the honey bee, and of dendrotoxin (DTX), a toxin from the green mamba snake, were studied in voltage-clamp experiments with myelinated nerve fibres of Xenopus. 2. MCDP and DTX blocked part of the K+ current. About 20% of the K+ current, however, was resistant to the toxins even in high concentrations. In Ringer solution half-maximal block was reached with concentrations of 33 nM-MCDP and 11 nM-DTX. In high-K+ solution the potency of both toxins was lower. beta-Bungarotoxin (beta-BuTX), another snake toxin, also blocked part of the K+ current, but was less potent than MCDP and DTX. 3. Tail currents in high-K+ solution were analysed and three K+ current components were separated according to Dubois (1981 b). Both MCDP and DTX selectively blocked a fast deactivating, slowly inactivating K+ current component which steeply activates between E = -60 mV and E = -40 mV (component f1). In concentrations around 100 nM, MCDP and DTX blocked neither the slow K+ current (component s) nor the fast deactivating, rapidly inactivating K+ current which activates between E = -40 mV and E = 20 mV (component f2). Similar results could be derived from K+ outward currents in Ringer solution. In high-K+, IC50 of MCDP for component f1 was 99 nM, whereas it was 7.6 microM for f2. Corresponding values for DTX are 68 nM and 1.8 microM. 4. Binding studies with nerve fibre membranes of Xenopus reveal high-affinity binding sites for 125I-labelled DTX (KD = 22 pM in Ringer solution and 81 pM in high-K+ solution). 125I-labelled DTX can be displaced from its sites completely by unlabelled DTX, toxin I (black mamba toxin), MCDP, and partially by beta-BuTX. 5. Immunocytochemical staining demonstrates that binding sites for DTX are present in nodal and paranodal regions of the axonal membrane. 6. The axonal membrane of motor and sensory nerve fibres is equipped with three types of well-characterized K+ channels and constitutes so far the best preparation to study MCDP- and DTX-sensitive K+ channels with electrophysiological and biochemical methods.

Crystals of Fasciculin 2 from Green Mamba Snake Venom: Preparation and preliminary x-ray analysis

Fasciculin 2 from the venom of the green mamba, Dendroaspis angusticeps, has been crystallized. The crystals are tetragonal, with unit cell dimensions a = 48.9 A and c = 82.0 A, space group P 41 21 2 or P 43212. Density measurements and pseudocentering of the hko zone indicate that there are 16 molecules in the unit cell.

Neurochemical and behavioral correlates of unilateral striatal acetylcholinesterase inhibition by fasciculin in rats

Fasciculin 2 (FAS) an anticholinesterase peptide isolated from the venom of the Green mamba ( Dendroaspis angusticeps) was injected into the right striatum of albino rats (1.5 μm total amount). The inhibition of acetylcholinesterase (AChE) activity was 86 and 60% 24 h and 7 days after FAS injection, respectively. The treatment with apomorphine (APO) (2 mg/kg s.c.) 24 h after FAS provoked a moderate circling towards the lesioned side that was reverted by atropine (30 mg/kg i.p.). The same dose of APO 7 days after FAS, provoked an inconstant contralateral circling. Neither dopamine nor serotonin not their metabolites were significantly affected 24 h or 7 days after FAS injection. Radioligand binding assays of dopamine, muscarinic and benzodiazepine receptors only showed a decrease of the density of the muscarinic ones 7 days after FAS. These results are interpreted as showing that the changes provoked by FAS would be compensated but the system would remain in an unsteady state only demonstrable after pharmacological challenge. The chronic down-regulation of muscarinic receptors would compensate the increased cholinergic activity and would therefore block its behavioral expression.

Effects of local inhibition of locus coeruleus acetylcholinesterase by fasciculin in rats

The inhibition of locus coeruleus (LC) acetylcholinesterase (AChE) by Fasciculin II (FAS), a novel anticholinesterase peptide from the green mamba ( Dendroaspis angusticeps) venom, was studied in rats. FAS was stereotaxically injected (0.5 μl of a 1 mg/ml solution) in the right LC. The left LC was taken as control. A group of rats received only saline injected with the same procedure. An inhibition of 80% of LC AChE activity was observed 24 h later. Monoamine and metabolite levels were assessed by high-performance liquid chromatography (HPLC) with electrochemical detection. A significant increase of noradrenaline (NA) levels was found in the injected side when compared with controls 24 h after injection. Neither dopamine, serotonin nor their metabolites or the NA metabolite 4-methoxyhydroxyphenylglycol showed any change after FAS injection. Atropine (30 mg/kg, i.p.) did not prevent the NA increase.

Two subtypes of acetylcholinesterase isoenzymes distinguishable by Angusticeps-type toxin F 7

1. Toxin F 7, a toxin isolated from Dendroaspis angusticeps (green mamba) venom, exhibited a potent inhibition on the acetylcholinesterase of Bungarus snake venoms and homogenized brains and muscles of Bungarus and Trimeresurus snakes as well as that of mammalian tissues. 2. The acetylcholinesterase in the venoms and tissues of Naja (cobra) species as well as that in avian tissues, however, was found to be about 1000 times less susceptible towards inhibition by toxin F 7. 3. It is concluded that there exist at least two subtypes of acetylcholinesterase isoenzymes distinguishable by an angnsliceps-type toxin F 7.

Cardiotoxicity of kenyan green mamba (dendroaspis angusticeps) venom and its fractionated components in primary cultures of rat myocardial cells

The cardiotoxic actions of Kenyan green mamba ( Dendroaspis angusticeps) venom have been investigated using primary myocardial cell cultures isolated from neonatal rat hearts. The cardiotoxic actions of the whole venom and its fractionated components were evaluated on the basis of leakage of lactate dehydrogenase (LDH), changes in morphology, cell membrane lysis, decreases in viability and inhibition of spontaneous beating activity. The whole venom caused time- and concentration-dependent arrest of myocardial contraction, leakage of LDH, extensive disruption of cell monolayer, and decreases in viability. The venom was separated into 6 (DaI to DaVI) fractions by gel permeation chromatography on Sephadex G-50. Spontaneous beating activity was abolished by DaI to DaVI at high concentrations, while at lower doses they induced progressive depression of beating frequency after a 3-h treatment period. DaI to DaIV caused significant leakage of LDH, morphological damage, and decreases in viability after a 6-h incubation period. The most cardiotoxic fraction (DaIV), which also contains about 54% of the total protein of the whole venom, was fractionated into 18 polypeptides (Da1 to Da18) by ion exchange chromatography on Bio-Rex 70. On the basis of their ability to abolish myocardial contractility, release LDH, alter cellular structure, lyse cell membranes and reduce viability, the 18 fractions have been divided into 4 arbitrary subgroups of cytotoxins: cardiotoxins, Da1 to Da3; cardiotoxin-like polypeptides, Da4 to Da12, Da14; less active membrane lytic polypeptides, Da13, Da15 to Da17; and membrane lytic polypeptide, Da18. Marked synergistic cell membrane lysis occurred in myocardial cell cultures treated simultaneously with 2 cardiotoxin-like polypeptides, Da7 and Da11. It is suggested that the additive and synergistic cardiotoxic effects of high molecular weight cytotoxic proteins (DaI to DaIII), very low molecular weight cholinomimetic substances (DaV to DaVI) and the 4 subgroups of cardiotoxins may directly contribute to the pronounced cardiovascular problems observed in victims of green mamba bites.

Toxins from the venom of the green mamba Dendroaspis angusticeps that inhibit the binding of quinuclidinyl benzilate to muscarinic acetylcholine receptors

Two protein toxins that displace the muscarinic antagonist quinuclidinyl benzilate from rat cortex synaptosomal membranes have been isolated from the green mamba ( Dendroaspis angusticeps) venom by gel filtration on sephadex G-50, chromatography on the ion-exchangers Bio-Rex 70 and Sulphopropyl-Sephadex C-25 and reversed-phase HPLC. Toxin 1 has 64 amino acids and four disulfides and a formula weight of 7200 and the corresponding values for toxin 2 are 63, 4 and 6840, respectively. Ultracentrifugation gave a molecular weight of 6900 for toxin 1 and 6700 for toxin 2, Quinuclidinyl benzilate that binds to all types of muscarinic cholinergic receptor was displaced to about 50% by both toxins. This partial displacement indicates that the toxins might be specific for one subtype of receptor.