Françoise Bouet

Conformational changes in two neurotoxic proteins from snake venoms.

alpha-Neurotoxin from Naja nigricollis and erabutoxin b from Laticauda semifasciata, two homologous neurotoxic proteins, are studied by circular dichroism, ultraviolet spectroscopy and fluorescence in various water/trifluoroethanol mixtures. The data obtained show that the beta structure of alpha-neurotoxin is conserved in water as well as in the organic solvent. By contrast, erabutoxin b changes from the beta-structure in water to the helix type in trifluoroethanol. The latter induces similarly for both toxins a structural modification around tryptophan 29, a residue common to all neurotoxins known to date. The vicinity of tyrosine 25, another common amino acid, is also altered by the presence of the organic solvent as demonstrated by the sudden increase of reactivity of the phenolic ring towards iodine. The present work affords some evidence for the presence of a particular structure located around the two aromatic residues, which is common to all neurotoxins and able to rearrange independently from the rest of the molecule. Biological importance of this peculiar region is highly probable.

Purification of macrophage migration inhibitory factor (MIF) from bovine brain cytosol.

Two isoforms of the macrophage migration inhibitory factor (MIF) have been isolated to homogeneity from bovine brain cytosol. In agreement with the cDNA sequence of their human counterpart, they both have an apparent molecular weight of 12 kDa and are characterized by the following N‐terminal amino acid sequence NH2‐PMFVVNTNVPRASVPDGLLSELTQQLAQATGKPPQYIAV‐. CD spectra revealed that bovine MIF contains 42% (±3%) α‐helix and 21% (±3%) β‐structure. CD‐constrained prediction of the secondary structure assigned MIF to the α/β‐class of proteins.

A new conotoxin isolated from Conus consors venom acting selectively on axons and motor nerve terminals through a Na+‐dependent mechanism

A novel conotoxin was isolated and characterized from the venom of the fish‐hunting marine snail Conus consors. The peptide was identified by screening chromatography fractions of the crude venom that produced a marked contraction and extension of the caudal and dorsal fins in fish, and noticeable spontaneous contractions of isolated frog neuromuscular preparations. The peptide, named CcTX, had 30 amino acids and the following scaffold: X11CCX7CX2CXCX3C. At the frog neuromuscular junction, CcTx at nanomolar concentrations selectively increased nerve terminal excitability so that a single nerve stimulation triggered trains of repetitive or spontaneous synaptic potentials and action potentials. In contrast, CcTx had no noticeable effect on muscle excitability even at concentrations 100 × higher than those that affected motor nerve terminals, as revealed by direct muscle stimulation. In addition, CcTx increased miniature endplate potential (MEPP) frequency in a Ca2+‐free medium supplemented with ethylene glycol‐bis‐(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid (EGTA). Blockade of voltage‐dependent sodium channels with tetrodotoxin (TTX) either prevented or suppressed the increase of MEPP frequency induced by the toxin. CcTx also produced a TTX‐sensitive depolarization of the nodal membrane in single myelinated axons giving rise, in some cases, to repetitive and/or spontaneous action potential discharges. In addition, CcTx increased the nodal volume of myelinated axons, as determined using confocal laser scanning microscopy. This increase was reversed by external hyperosmolar solutions and was prevented by pretreatment of axons with TTX. It is suggested that CcTx, by specifically activating neuronal voltage‐gated sodium channels at the resting membrane potential, produced Na+ entry into nerve terminals and axons without directly affecting skeletal muscle fibres. CcTx belongs to a novel family of conotoxins that targets neuronal voltage‐gated sodium channels.

Neuromuscular effects of some potassium channel blocking toxins from the venom of the scorpion Leiurus quinquestriatus hebreus

The scorpion venom Leiurus quinquestriatus hebreus was fractionated by chromatography in order to isolate toxins that affected binding of radiolabelled dendrotoxin to K+ channel proteins on synaptosomal membranes and that facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations. In addition to the previously characterized charybdotoxin, three toxins were isolated: 14-2, 15-1 and 18-2. Toxin 14-2 has a blocked N-terminus and because of low quantities, it has not been sequenced; 15-1 is a newly sequenced toxin of 36 residues with some overall homology to charybdotoxin and noxiustoxin; 18-2 is identical to charybdotoxin-2. The apparent Ki against dendrotoxin binding were: charybdotoxin, 3.8 nM; 14-2, 150 nM; 15-1, 50 nM; and 18-2, 0.25 nM. Toxin 14-2 (75 nM-1.5 microM) had a presynaptic facilitatory effect on neuromuscular preparations. Toxin 15-1 augmented responses to direct muscle stimulation, probably because it blocked Ca(2+)-activated K+ currents in muscle fibres. Toxin 18-2 (charybdotoxin-2) had a potent presynaptic facilitatory action, with less effect on direct muscle stimulation. This contrasts with the relatively weak neuromuscular effects of the highly homologous charybdotoxin. On a Ca(2+)-activated K+ current in mouse motor nerve endings, charybdotoxin and toxin 18-2 produced maximal block at around 100 nM, whereas 15-1 was inactive at 300 nM. Charybdotoxin can increase quantal content, but this is more likely to result from block of voltage-dependent K+ channels than Ca(2+)-activated channels: the increase in transmitter release occurred in conditions in which little IKCa would be present; higher concentration of charybdotoxin and longer exposure times were required to increase transmitter release than those needed to block IKCa, and the facilitatory effects of charybdotoxin and toxin 18-2 correlated more with their effects on dendrotoxin binding than on block of IKCa.

Cyclophilin-B is an abundant protein whose conformation is similar to cyclophilin-A.

Cyclophilin‐B (bCyP‐20) was isolated in a relatively high quantity from calf brain and spleen tissues consecutively applying weak cation exchange, chromatofocusing and strong cation exchange chromatographies. Edman degradation yielded the N‐terminal sequence NH2‐DEKKKGPKVTVK‐VYFDLRIGDEDIGRVVIGLFGKTVPKTVDNFVAL. Bovine cyclophilin‐B possesses the peptidylproline cis‐trans isomerase activity which is inhibited by nM concentrations of CsA. bCyP‐20 has a strong tendency to bind to cation exchangers including DNA and heparin. It could be released from DNA affinity column at concentrations of NaCl higher than 200 mM. Circular dichroism spectroscopy revealed that bovine cyclophilin‐A (bCyP‐18) and bCyP‐20 in aqueous solution have similar conformations.

On the purification of notexin: Isolation of a single amino acid variant from the venom of Notechis scutatus scutatus

Venom of the Australian tiger snake, Notechis scutatus scutatus was fractionated by conventional ion‐exchange chromatography. The fraction containing notexin, a well‐known single‐chain toxic phospholipase A2, was further purified by reverse‐phase high‐performance liquid chromatography. Two main components were isolated and the major one corresponded to notexin. The other component, designated as notechis Ns, was an isofonn of notexin. Notechis Ns and notexin possessed similar in vitro esterase activity, in vitro neuromuscular activity and in vivo lethality. Amino acid composition and sequence of the Staphylococcus aureus V8‐protease peptides demonstrated that primary structures of notechis Ns and notexin differed from each other by a single substitution amongst 119 amino acids: Lys → Arg at position 16.

Structural Analysis of Acetylcholinesterase Ammonium Binding Sites

The identification of the amino acids residues belonging to acetylcholinesterase (AchE) binding sites has been first undertaken through physicochemical determinations or by means of chemical modification experiments. In particular, Trp, Tyr, His and Lys residues have been suggested to be involved in such interactions (Shinitzky et al., 1973; Majumdar and Balasubramanian, 1984; Fuchs et al., 1984; Page and Wilson, 1985). Later, site directed labelling experiments using photoaffinity (Kieffer et al., 1986) and affinity (Weise et al., 1990) probes, showed respectively that a tetrapeptide Gly-Ser-X-Phe (corresponding to amino acids 328 to 331 in Torpedo AchE, X is the labelled residue) and Trp84 belong to the quaternary ammonium binding site of the enzyme.

Etude moleculaire de neurotoxines de venins de serpents marins et terrestres

The present paper describes (1) Biosynthesis of a snake neuro— toxic protein and refolding of its polypeptide chain; (2) Identification of the surfaces by which a neurotoxin interacts with: i) the acetylcholine receptor, ii) specific monoclonal antibodies.

Studies of tubocurarine labelled with iodine or tritium

Summary D-tubocurarine was reacted first with ICl and then with tritium by catalytic hydrogenolysis of the iodo derivative in the presence of tritium gaz. The specific radioactivity of the tritiated product reached 12 Ci/mmole and its biological activity was identical to that of the original material. From the iodinated material (0.8 atom per mole) pure monoiodo-d-tubocurarine was obtained by selective adsorption on a Biogel column. Localization of the halogen was characterized by pKs measurements of the two phenolic functions of the iodo and of the original d-tubocurarine. Circular dichroism spectra and toxicity measurements of the iodinated compound have shown that iodine does not, or very slightly only, modify the structural and biological properties of d-tubocurarine.