Improved method for the isolation, characterization and examination of neuromuscular and toxic properties of selected polypeptide fractions from the crude venom of the Taiwan cobra Naja naja atra.

An improved chromatographic method was developed to isolate and purify polypeptides and proteins from the crude venom of the Taiwan cobra Naja naja atra. The procedure devised is simple, easy to reproduce, and enables large scale isolation of almost all polypeptides and proteins in this cobra venom. Six pure polypeptide fractions of the venom were isolated and characterized using gel filtration on Sephadex G50 (medium), ion exchange chromatography on SP-Sephadex C25, desalting on Sephadex G25 (fine) and preparative HPLC on a RPC 18 column. The neuromuscular activity of these fractions was tested on the chick biventer cervicis nerve-muscle preparation and their toxicity (LD(50)) was determined after i.v. administration in mice. Their antinociceptive activity was tested in the mouse abdominal test by i.v. application. Two of these polypeptide samples had major physiological effects: one acted as a cardiotoxin causing reversible myocardial contractures with no effect on muscle twitches elicited by nerve stimulation (NS); another was a neurotoxin that blocked muscle contractions in response to NS and exogenously added acetylcholine. The cardiotoxic fraction was identified as CTX I, a well-known cardiotoxin present in this venom, and the neurotoxin was identified as neurotoxin-α with an LD50 in mice of 0.075 mg/kg.

[Haemostaseologic effects of fractionated snake venoms]. / Wirkungen von Schlangengiftfraktionen auf das Gerinnungssystem.

Comparative investigation concerning gelfiltration as well as haemostaseologic analysis of venoms and venom fractions of some snakes (elapidae and viperidae) have shown that in elapidae an inhibition of coagulation is dominant whilst in viperidae the stimulation of coagulation is of importance. Our investigations produce a basis to select substances for activation of coagulation and substances for inhibition of coagulation. Under pharmacological viewpoints the data may produce information to use snake fractions for anticoagulation or for procoagulant therapy in bleeding tendency.

Isolation and characterisation of five neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima.

Five toxins (APE 1 to APE 5) of the sea anemone species Anthopleura elegantissima (Brandt) have been isolated from a toxic by-product fraction of its concentrated crude watery-methanolic extract, prepared previously for the isolation of a neuropeptide (the head-activator) by Schaller and Bodenmüller (Proc. Natl. Acad. Sci. USA 78 (1981) 7000) from 200kg sea anemones. Toxin purification was performed by desalting of the starting material by dialysis (MWCO 3500) against distilled water, anion exchange chromatography on QAE-Sephadex A25 at pH 8, twice gel filtration on Sephadex G50 m, repeated chromatography on QAE-Sephadex at pH 10 and chromatography on the cation exchanger Fractogel EMD SO(3)(-)-650 M.Final purification of the toxins was achieved by HPLC on MN SP 250/10 Nucleosil 500-5 C(18) PPN and MN SP 250/21 Nucleosil 300-7 C(18). Each toxin was composed of at least two isotoxins of which APE 1-1, APE 1-2, APE 2-1, APE 2-2 and APE 5-3 were isolated in preparative scale. With exception of APE 5-3 the sequences of the isotoxins have been elucidated. They resemble the 47 residue type-I long polypeptide toxins native to Anemonia sulcata (Pennant). All isotoxins paralyse the shore crab (Carcinus maenas) by tetanic contractions after i.m. application. The toxins modify current passing through the fast Na(+) channel in neuroblastoma cells, leading to delayed and incomplete inactivation. APE 1-1, APE 2-1 and APE 5-3 produce a positive inotropic effect in mammalian heart muscle, although they differ in potency. The order of potency is APE 2-1>APE 1-1>APE 5-3 (i.e. threshold concentrations are 1, 10 and 300nM, respectively). In addition, they enhance the spontaneous beating frequency in isolated right atria (guinea pig). The most potent cardiotoxic isotoxin is APE 2-1, its sequence is identical with that of AP-C, a toxin isolated and characterised previously by Norton et al. (Drugs and Foods from the Sea, 1978, University of Oklahoma Press, p. 37-50).LD50 APE 2-1:1 micro g/kg b.w. C. maenas (i.m.). LD50 APE 1-1:10 microg/kg b.w. C. maenas (i. m.). LD50 APE 5-3:50 microg/kg b.w. C. maenas (i.m.).

Sea anemone peptides with a specific blocking activity against the fast inactivating potassium channel Kv3.4.

Sea anemone venom is known to contain toxins that are active on voltage-sensitive Na+ channels, as well as on delayed rectifier K+ channels belonging to the Kv1 family. This report describes the properties of a new set of peptides from Anemonia sulcata that act as blockers of a specific member of the Kv3 potassium channel family. These toxins, blood depressing substance (BDS)-I and BDS-II, are 43 amino acids long and differ at only two positions. They share no sequence homologies with other K+ channel toxins from sea anemones, such as AsKS, AsKC, ShK, or BgK. In COS-transfected cells, the Kv3.4 current was inhibited in a reversible manner by BDS-I, with an IC50 value of 47 nM. This inhibition is specific because BDS-I failed to block other K+ channels in the Kv1, Kv2, Kv3, and Kv4 subfamilies. Inward rectifier K+ channels are also insensitive to BDS-I. BDS-I and BDS-II share the same binding site on brain synaptic membranes, with K0.5 values of 12 and 19 nM, respectively. We observed that BDS-I and BDS-II have some sequence homologies with other sea anemone Na+ channels toxins, such as AsI, AsII, and AxI. However, they had a weak effect on tetrodotoxin-sensitive Na+ channels in neuroblastoma cells and no effect on Na+ channels in cardiac and skeletal muscle cells. BDS-I and BDS-II are the first specific blockers identified so far for the rapidly inactivating Kv3.4 channel.

Anticoagulant fucoidan fractions from Fucus vesiculosus induce platelet activation in vitro.

Anticoagulant fucoidan fractions of different molecular weight and sulfate content were prepared and investigated for their effects on platelet function in vitro. The fucoidan fractions were incubated with human platelet rich plasma (PRP) at concentrations of 5, 10 and 50 micrograms/ml. Platelet activation was subsequently studied by a standard aggregation assay and flow cytometric determination of the activation dependent platelet-surface markers CD62p (P-selectin, GMP-140) and CD63 (GP53). All fucoidan fractions induced irreversible platelet aggregation in a dose-dependent manner. Comparing fractions of identical molecular weight (100 kDa) the low sulfate content fucoidan FF5 (S = 7.6%) exerted a significantly greater effect than the highly sulfated fucoidan FF7 (S = 10.2%) over the whole concentration range (n = 5, P < 0.05). Among fractions of identical sulfate content fucoidan-induced platelet aggregation was also found to depend on the molecular weight of the fucoidan. At concentrations of 10 and 50 micrograms/ml the high molecular weight fraction FF7/1 (150 kDa) showed a significantly greater effect than the 50 kDa fraction FF7/3 (24.8 +/- 6.7 vs. 7.0 +/- 3.5 and 54.6 +/- 13.5 vs. 15.0 +/- 9.0%, respectively; mean +/- SD, n = 5, P < 0.05). The molecular weight dependence of the fucoidan effect was also reflected by the flow cytometric data. Coincubation of FF7/1 and FF7/3 (10 micrograms/ml) with PRP increased the number of CD62p and CD63 positive platelets by 9.0 +/- 3.3 vs. 2 +/- 1.9 and 7.1 +/- 2.4 vs. 3.2 +/- 2.6% over control values, respectively (n = 5, P < 0.05). In conclusion, our results show that the low molecular weight fucoidan FF7/3 combines potent anticoagulant and fibrinolytic properties with only minor platelet activating effects and is therefore a suitable substance for further pharmacological studies.

A simple biochemical method in the search for bioactive polypeptides in a sea anemone (Anemonia sulcata).

The sea anemone Anemonia sulcata is a well-known natural source of supply of biologically active polypeptides. So far, five toxins, ATX I, II, III, IV and AS V, several polyvalent protease inhibitors, an elastase inhibitor, two blood pressure-depressive polypeptides and very recently peptides that inhibit competitively the binding of 125I-dendrotoxin to rat brain membranes and block the voltage-sensitive K+ channels, have been isolated from it. The sea anemone toxins (especially toxin II of A. sulcata, ATX II) are very important tools in neurophysiological and pharmacological research, and their structure-function relationship has been investigated. Because of the great scientific value of the sea anemone toxins a simplification of their purification procedure was elaborated.

Kalicludines and kaliseptine. Two different classes of sea anemone toxins for voltage sensitive K+ channels.

New peptides have been isolated from the sea anemone Anemonia sulcata which inhibit competitively the binding of 125I-dendrotoxin I (a classical ligand for K+ channel) to rat brain membranes and behave as blockers of voltage-sensitive K+ channels. Sea anemone kalicludines are 58-59-amino acid peptides cross-linked with three disulfide bridges. They are structurally homologous both to dendrotoxins which are snake venom toxins and to the basic pancreatic trypsin inhibitor (Kunitz inhibitor) and have the unique property of expressing both the function of dendrotoxins in blocking voltage-sensitive K+ channels and the function of the Kunitz inhibitor in inhibiting trypsin. Kaliseptine is another structural class of peptide comprising 36 amino acids with no sequence homology with kalicludines or with dendrotoxins. In spite of this structural difference, it binds to the same receptor site as dendrotoxin and kalicludines and is as efficient as a K+ channel inhibitor as the most potent kalicludine.

Structure of cobra cardiotoxin CTX I as derived from nuclear magnetic resonance spectroscopy and distance geometry calculations.

The structure of cardiotoxin CTX I from Naja naja atra has been investigated by NMR spectroscopy. Sequence specific resonance assignments have been obtained for all backbone protons as well as for most side-chain protons. Distance geometry calculations were carried out using a metric matrix DG program. A total of 715 NOE constraints, 27 phi angle constraints and a list of the hydrogen bond donors were used for the metric matrix DG calculations and refinement. The average pairwise r.m.s.d. of the resulting structures was 1.01 A for the backbone heavy atoms, and 1.69 A for all heavy atoms. The protein is rich in beta structure and consists of a large triple-stranded, antiparallel beta sheet as well as a short double-stranded, antiparallel beta sheet. Non-regular hydrogen bonding is found between side-chains of the carboxy-terminal end and the rest of the core region. The structure is discussed in terms of evolutionary aspects as well as recent investigations about the biological function and active site.

A new procedure for the isolation of anti-HIV compounds (polysaccharides and polyphenols) from the marine alga Fucus vesiculosus.

Anti-HIV-active polysaccharides and polyphenols were isolated from the brown seaweed Fucus vesiculosus by hot H2O extraction of both the intact and the homogenized algae. This was followed by XAD2 chromatography and by sequential precipitation of the non-adsorbed compounds with glacial HOAc and thereafter with EtOH. The precipitate was solubilized, dialyzed against distilled H2O, and chromatographed on SP-Sephadex C25 and on QAE-Sephadex A25. This was followed by gel filtration on Sephadex G50 and Sephadex G100 and finally by hplc on a Shodex Ionpak S-804 column. For comparison, the commercial product fucoidan, a sulfated algal polysaccharide, was also further purified by the chromatographic techniques mentioned above. The isolated freeze-dried fractions obtained by these procedures were tested for inhibition of both HIV-induced syncytium formation and HIV reverse transcriptase enzyme activity. Some of these fractions inhibited both of these activities at concentrations that were not cytotoxic.

Specificity of antibodies to sea anemone toxin III and immunogenicity of the pharmacological site of anemone and scorpion toxins.

Toxin III (ATX III) of the sea anemone (Anemonia sulcata) is a polypeptide containing 27 amino acid residues. It has no sequence similarity with other toxins (ATX I and II) from the same species, or with scorpion toxins, although they apparently act in a similar manner by prolonging action potentials. The specificity of ATX III antibodies was characterized using ATX III, ATX I, native and chemically modified ATX II, and scorpion alpha-toxins. The results obtained suggest that a region of ATX III, partially or totally overlapping the pharmacological site shared with ATX I and ATX II, is immunogenic. It includes a guanidino and at least two carboxylate groups. The corresponding region is not immunogenic in ATX I and ATX II. Anti-(ATX III) antibodies recognize the similar regions of ATX I and ATX II and apparently do not recognize scorpion toxins.

A proton nuclear magnetic resonance study of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: sequential and stereospecific resonance assignment and secondary structure.

The sequential resonance assignment of the 1H NMR spectrum of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata is presented. This is carried out with two-dimensional NMR techniques to identify through-bond and through-space (less than 5 A) connectivities. Added spectral complexity arises from the fact that the sample is an approximately 1:1 mixture of two BDS-I isoproteins, (Leu-18)-BDS-I and (Phe-18)-BDS-I. Complete assignments, however, are obtained, largely due to the increased resolution and sensitivity afforded at 600 MHz. In addition, the stereospecific assignment of a large number of beta-methylene protons is achieved from an analysis of the pattern of 3J alpha beta coupling constants and the relative magnitudes of intraresidue NOEs involving the NH, C alpha H, and C beta H protons. Regular secondary structure elements are deduced from a qualitative interpretation of the nuclear Overhauser enhancement, 3JHN alpha coupling constant, and amide NH exchange data. A triple-stranded antiparallel beta-sheet is found to be related to that found in partially homologous sea anemone polypeptide toxins.

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing.

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.

Natural proteinase inhibitors: blood coagulation inhibition and evolutionary relationships.

1. Natural proteinase inhibitors are divided into polysaccharides, plasma proteinase inhibitors and natural non-plasma inhibitors. 2. Polysaccharides are antithrombin-III and heparin co-factor-II dependent or independent regarding their biological activity. Knowledge of the inhibitory mechanism at a molecular level was gained by the study of heparin. 3. Antithrombin-III, heparin-co-factor-II and alpha 2-macroglobulin are the most important plasma proteinase inhibitors involved in coagulation. alpha 2-macroglobulin has a particular inhibitory mechanism. 4. Non-plasma proteinase inhibitors were isolated from many species. They inhibit mainly the contact activation and fibrinolysis. 5. The evolutionary relationships are poorly understood.

Immunochemistry of sea anemone toxins: structure-antigenicity relationships and toxin-receptor interactions probed by antibodies specific for one antigenic region.

Two antibody subpopulations directed against Anemonia sulcata toxin I or II have been purified by immunoaffinity chromatography. These antibodies are specific for a single antigenic region and were used in a structure-antigenicity relationship study using homologous toxins and chemically modified derivatives of A. sulcata toxin II. Asp-7 and/or Asp-9 and Gln-47 of toxin II were found to be implicated in the antigenic region recognized by the two antibody subpopulations. On the contrary, Arg-14, Lys-35, -36, and -46, and alpha-NH2 of the glycine residue of A. sulcata toxin II are not involved in the corresponding antigenic region. When assayed for interaction with the sodium channel, the antigenic region of toxin II, including Asp-9 and Gln-47, appeared fully accessible to its specific antibodies, suggesting that it is not involved in the binding of the toxin to its receptor.

Involvement of (Na+ + K+)-ATPase in binding and actions of palytoxin on human erythrocytes.

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The Kd from velocity and equilibrium measurements is 2 X 10(-11) M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) and by borate. It is inhibited by K+ (IC50 2 mM), ouabain (IC50 3 X 10(-9) M) and ouabagenin (IC50 6 X 10(-6) M). Conversely, [3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin (Ki 3 X 10(-11) M). Dog erythrocytes, which are known to possess a very low (Na+ + K+)-ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [3H]ouabain, depends on the state of (Na+ + K+)-ATPase. ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg2+ and Pi. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) (Na+ + K+)-ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through (Na+ + K+)-ATPase or its close vicinity.

Photochemically induced dynamic nuclear polarisation NMR study of the aromatic residues of sea-anemone polypeptide cardiac stimulants.

One-dimensional and two-dimensional photochemically induced dynamic nuclear polarisation (photo-CIDNP) nuclear magnetic resonance spectra have been recorded for the sea-anemone polypeptide cardiac stimulants anthopleurin-A and Anemonia sulcata toxins I and II. In anthopleurin-A and toxin II, all three Trp residues are accessible to the flavin dye, although Trp-23 in anthopleurin-A shows a weaker photo-CIDNP response than Trp-33 and Trp-45. Tyr-25 in anthopleurin-A also shows a strong response. In toxin I, Trp-23, Trp-33 and Tyr-45 (which replaces Trp in this molecule) are accessible to the dye. The pH dependences of the photo-CIDNP spectra of all three polypeptides have been examined. The response of Trp-33 increases significantly with pH. The two His residues of anthopleurin-A and toxin II display a response in their imidazole forms, but not their imidazolium forms. The surface accessibilities of Trp-23 and Trp-33 are discussed in relation to the interaction of these polypeptides with the Na+ channel.

Assignment of aromatic resonances in the 1H nuclear magnetic resonance spectra of cardioactive polypeptides from sea anemones.

High-resolution 1H NMR spectroscopy at 300 MHz has been used to investigate the aromatic residues of a series of homologous polypeptides from sea anemones: anthopleurin-A from Anthopleura xanthogrammica and toxins I and II from Anemonia sulcata. Using two-dimensional NMR techniques, specific assignments to individual protons have been made for all aromatic resonances in the spectra of these molecules. In all three polypeptides the resonances from the two conserved Trp residues, 23 and 33, are shifted significantly from their random coil values, and the indole NH resonance of Trp-23 is not observed. These shift perturbations are due in part to a mutual interaction of the two indole rings, which is also indicated by the observation of nuclear Overhauser enhancements between protons of the two rings. Several other nonpolar side chains also interact with these two Trp residues, forming a hydrophobic region, the overall structure of which is conserved throughout the series. The other aromatic residues in these polypeptides appear not to participate in this structural region.

Palytoxin-induced permeability changes in excitable membranes.

Palytoxin, a toxin isolated from the Caribean corrall Palythoa caribaeorum, increases the cation permeability of excitable membranes in vitro. Three membrane systems have been investigated: axonal membranes from crayfish walking leg nerves, membranes rich in nicotinic acetylcholine receptor isolated from Torpedo californica electric tissue and, for control, artificial liposomes. Ion permeability of the latter was not affected by palytoxin, but with both biological membranes an increase in cation permeability was observed at a palytoxin concentration of 0.14 microM. Palytoxin-induced cation flow through the axonal membrane was not inhibited by tetrodotoxin, indicating that the voltage-dependent sodium channels were not involved. The effect of palytoxin on the receptor-rich membranes was not blocked by alpha-bungarotoxin, a competitive antagonist of the nicotinic acetylcholine receptor, nor by triphenylmethylphosphonium, a blocker of the receptor-ion channel. But with both the axonal and the receptor-rich membranes ouabain was an inhibitor of the palytoxin-induced cation flow. Evidence is presented that it is not the (Na+ + K+)-ATPase which is affected by palytoxin as has been postulated for similar observations with non-neuronal membranes (Chhatwal, G.S., Hessler, H.-J. and Habermann, E. (1983) Naunyn-Schmiedeberg's Arch. Pharmacol. 323, 261-268).