Radioiodination of scorpion and snake toxins

Rochat, Hervé; Tessier, M.; Miranda, F.; Lissitzky, Serge

doi:10.1016/0003-2697(77)90192-0

Cited by 99 publications

(36 citation statements)

References 21 publications

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“…Fractions were collected and analyzed for radioactivity: 95 f 5% of the immuno-precipitated iodinated NmmI contained "'I-Tyr. So, as a result of the specific radioactivity obtained, it was concluded that NmmI was monoiodinated on Tyr-35, as previously reported [18].…”

Section: Lodination Of the A-neurotoxinsupporting

confidence: 83%

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Selective distinction at equilibrium between the two α‐neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

Marchot

Frachon

Bougis

1988

European Journal of Biochemistry

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The binding of the monoiodinated a-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kdl = 7 4 pM and Kd2 = 51 & 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-l,l = 25 x l o p 6 s-' and k -l . 2 = 623x s-l respectively), but not in their rate of formation of the toxin-receptor complex ( k + , = 11.7 x lo6 M-' S-' ). From these rate constants the same two values of dissociation constant were deduced (&I = 2 pM and Kdz = 53 pM). All the specific binding was prevented by the cholinergic antagonists abungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 pM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and a-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short a-neurotoxin are selectively distinguishable.

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Section: Lodination Of the A-neurotoxinsupporting

confidence: 83%

“…NmmI was enzymatically iodinated with 251 (Amersham) using lactoperoxidase (Sigma) and recovered by immunopre-cipitation [18]. A specific radioactivity of 1500 Ci/mmol was routinely achieved.…”

Section: Lodination Of the A-neurotoxinmentioning

confidence: 99%

Selective distinction at equilibrium between the two α‐neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

Marchot

Frachon

Bougis

1988

European Journal of Biochemistry

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“…His-Css4 was radioiodinated by lactoperoxidase (Sigma; Cat. No., L8257; 7 units/60 l reaction mix) using 10 g of toxin and 0.5 mCi of carrier-free Na 125 I (Amersham Biosciences) following a previously published protocol (21). The monoiodotoxin was purified using an analytical resource reverse phase-HPLC 1 column (4.6 ϫ 100 mm, 15-m particle size; Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Common Features in the Functional Surface of Scorpion β-Toxins and Elements That Confer Specificity for Insect and Mammalian Voltage-gated Sodium Channels

Cohen

Karbat

Gilles

et al. 2005

Journal of Biological Chemistry

125

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Scorpion ␤-toxins that affect the activation of mammalian voltage-gated sodium channels (Na v s) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion ␤-toxin, Css4, whose effects on rat brain Na v s have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 Å 2 composed of the following: 1) a cluster of residues associated with the ␣-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion ␤-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the ␤2 and ␤3 strands, which is likely to confer the specificity for mammalian Na v s; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion ␤-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.

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“…AaH I, AaH I1 and AaH 111 were iodinated by the lactoperoxidase method of [' 251]iodide oxidation and were purified by immunoprecipitation with antisera prepared against AaH I, AaH I1 and AaH 111, respectively, according to Rochat et al [16]. The specific radioactivities were 780, 800 and 750 Ci/mmol, respectively.…”

Section: Chemical Modificationsmentioning

confidence: 99%

Structure/activity relationships of scorpion α‐toxins

Kharrat

Darbon

Rochat

et al. 1989

European Journal of Biochemistry

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Chemical modifications of tyrosine and tryptophan residues of scorpion a-neurotoxins I1 and I11 from Androctonus australis Hector were performed as well as modification of the two arginines and the a-amino group of toxin I. The pharmacological potencies of each derivative were assessed in vivo by LDS0 measurement and in vitro by competition experiments with '251-toxin for synaptosomal receptors. Arginine residues in positions 2 and 60 and the a-amino group of Androctonus toxin 1 were derivatized by p-hydroxyphenylglyoxal; the corresponding modified toxins exhibit low pharmacological potencies. Tryptophan 38 of toxin I1 and tryptophan 45 of toxin 111 were modified by nitrophenylsulfenyl chloride, leading respectively to a poorly and a fully active derivative. The tetranitromethane modification of tyrosine residues in positions 60, 5 and 14 of toxin 111 induced respectively 60%, 40% and 30% of loss of biological activity. Circular dichroic analysis indicated that for every derivative, except the nitrophenylsulfenyl derivative of Trp-45 of AaH 111, the conformation of the toxin was not altered by derivatization. Conformational integrity was also confirmed by full activity of the derivatives in radioimmunoassays. Taken together, the results suggest that aromatic residues belonging to the conserved hydrophobic surface, to the C-terminal and to the loop region 37-44 are involved in the molecular mechanisms by which scorpion atoxins act. Charged residues in the N-terminal and C-terminal also contribute to the high efficacy of the binding process. It appears that all important residues are clustered on one face of the toxin, suggesting a multipoint interaction with the proteins of the sodium channel.Scorpion toxins are known to interact specifically with the voltage-dependent sodium channel 11, 21. They form a family of structurally related proteins made of a single polypeptide chain and having a molecular mass of about 7 kDa (60 -70 amino acid residues) [3] (for a review on isolation and structure, see [4]). They can be divided into mammal and insect toxins according to their specificity toward mammalian and insect nervous systems [5]. Two types (a and p ) of mammal scorpion toxins have been described according to their pharmacological properties and their binding to two different sites [6]. These toxins act at the level of the voltage-dependent sodium channel in different ways. Thus a-toxins bind to the receptor in a potential-dependent manner whereas p-toxins do not; a-toxins induce a prolongation of the repolarization phase of the action potential while p-toxins promote a repetitive firing after a unique stimulation [7].The three-dimensional structures of a protein from the venom of Centruroides sculpturatus Ewing (CsE V,) [8] and of toxin I1 from the venom of Androctonus australis Hector (AaH 11) [9] were elucidated at high resolution by X-ray crystallography and may be taken as structural models for respectively P-and a-toxins. In both cases an a-helix structure and three short strands of P-sheet are found in simi...

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Radioiodination of scorpion and snake toxins

Cited by 99 publications

References 21 publications

Selective distinction at equilibrium between the two α‐neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

Selective distinction at equilibrium between the two α‐neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

Common Features in the Functional Surface of Scorpion β-Toxins and Elements That Confer Specificity for Insect and Mammalian Voltage-gated Sodium Channels

Structure/activity relationships of scorpion α‐toxins

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