Eliahu Zlotkin

An excitatory and a depressant insect toxin from scorpion venom both affect sodium conductance and possess a common binding site

Two insect selective toxins were purified by gel-permeation and ion-exchange chromatographies from the venom of the scorpion, Leiurus quinquestriatus quinquestriatus, and their chemical and pharmacological properties were studied. The first toxin (LqqIT1) induces a fast excitatory contraction paralysis of fly larvae and is about 40 times more toxic than the crude venom. It is a polypeptide composed of 71 amino acids, including 8 half-cystines and devoid of methionine and tryptophan, with an estimated molecular weight of 8189 and a pI value of 8.5. The second toxin (LqqIT2) induces a slow depressant, flaccid paralysis of fly larvae. It is composed of 72 amino acids, including 8 half-cystines, is devoid of proline methionine and histidine, and has an estimated molecular weight of 7990 and a pI value of 8.3. The contrasting symptomatology of these toxins is interpreted in terms of their effects on an isolated axonal preparation of the cockroach in current and voltage clamp conditions. LqqIT1 (0.5-4 microM) induced repetitive firing of the axon which was attributable to two changes in the sodium conductance, a small increase in the peak conductance and a slowing of its turning off. LqqIT2 (1-8 microM) caused a blockage of the evoked action potentials, attributable to both a strong depolarization of the axonal membrane and a progressive suppression of the sodium current. Neither toxin affected potassium conductance. The two toxins differ mainly in their opposite effects on the activatable sodium permeability. In binding assays to a preparation of insect synaptosomal membrane vesicles, the two toxins were shown to competitively displace the radioiodinated excitatory insect toxin derived from the venom of the scorpion, Androctonus australis [( 125I]AaIT), which strongly resembles, in its chemistry and action, the LqqIT1 toxin. The present two toxins have demonstrated a strong affinity closely resembling the AaIT, with KD values of 0.4, 1.9, and 1.0 nM for LqqIT1, LqqIT2, and AaIT, respectively. These data suggest the possibility that the excitatory and depressant insect toxins share a common binding site associated with sodium channels in insect neuronal membranes.

Purification and properties of the insect toxin from the venom of the scorpion Androctonus australis Hector

Summary The purification of a protein toxic to insects from the venom of the scorpion Androctonus australis Hector has been performed by recycling Sephadex G-50 gel filtration and equilibrium chromatography on DEAE-Sephadex A-50 and Amberlite CG-50. The final product was purified 267-fold as compared to the crude venom. The toxicity yield was 95 percent. Purity of the insect toxin was assessed by amino acid analysis, N-terminal sequential degradation, C-terminal amino acid determination and zone electrophoresis. Its molecular weight is 7498. The insect toxin differs from the proteins toxic to mammals contained in the same venom. The sequence of the first 15 amino acid residues from the N-terminal of both types of toxins is compared. The physiological significance of these neurotoxic proteins is discussed.

The effect of scorpion venom on blowfly larvae-a new method for the evaluation of scorpion venoms potency.

The injection of small amounts of scorpion venom in blowfly larvae causes an immediate contraction and paralysis, the duration of which is dosage dependent. This response of fly larvae, considered to represent a peripheral neurotoxic effect of scorpion venoms, is suggested as a rapid and sensitive test for the quantitative estimation of the potency of scorpion venoms and antisera. The contraction-paralysis of a minimal duration of 5 sec is defined as a standard positive response. The amount of venom causing 50 per cent positive responses is defined as a unit of toxicity and called the Contraction-Paralysis Unit (CPU). A comparison between mice ld50 and CPU of eighteen scorpion venoms demonstrates the absence of correlation between the two phenomena. This finding suggests the possibility that different neurotoxic components of scorpion venom are responsible for the toxic effects on mice and fly larvae.

AaIT: from neurotoxin to insecticide.

AaIT is a single chain neurotoxic polypeptide derived from the venom of the Buthid scorpion Androctonus australis Hector, composed of 70 amino acids cross-linked by four disulfide bridges. Its strict selectivity for insects has been documented by toxicity, electrophysiological and ligand receptor binding assays. These last have shown that various insect neuronal membranes possess a single class of non-interacting AaIT binding sites of high affinity (K(D) = 1-3(n)M) and low capacity (0.5-2.0 pmol/mg prot.). The fast excitatory paralysis induced by AaIT is a result of a presynaptic effect, namely the induction of a repetitive firing in the terminal branches of the insects motor nerves resulting in a massive and uncoordinated stimulation of the respective skeletal muscles. The neuronal repetitive activity is attributed to an exclusive and specific perturbation of sodium conductance as a consequence of toxin binding to external loops of the insect voltage-dependent sodium channel and modification of its gating mechanism. From a strictly agrotechnical point of view AaIT involvement in plant protection has taken the following two complementary forms: firstly, as a factor for the genetic engineering of insect infective baculoviruses resulting in potent and selective bio-insecticides. The efficacy of the AaIT-expressing, recombinant baculovirus is attributed mainly to its ability to continuously provide and translocate the gene of the expressed toxin to the insect central nervous system; secondly, based on the pharmacological flexibility of the voltage-gated sodium channel, as a device for insecticide resistance management. Channel mutations conferring resistance to a given class of insecticidal agents (such as the KDR phenomenon) may greatly increase susceptibility to the AaIT expressing bioinsecticides. Thus the AaIT is a pharmacological tool for the study of insect neuronal excitability and chemical ecology and the development of new approaches to insect control.

Purification, characterization and action of two insect toxins from the venom of the scorpion buthotus judaicus

Abstract Two toxic proteins, insect toxins I and II, selectively paralytic and lethal to insects, were purified from the venom of the Black scorpion, Buthotus judaicus , using gel permeation and ion-exchange chromatography. Their chemical purity and characteristics were assessed by column chromatography, disc electrophoresis, isoelectrofocusing and amino acid analyses. Their biological specificity and toxicity were determined by a series of paralysis and lethality assays employing the larvae of the flesh fly, Sarcophaga falculata , and the locust, Locust migratoria . Their modes of action were investigated employing in vitro cockroach axonal preparations in current- and voltage-clamp conditions. Insect toxin I (IT-I) is approximately 40-times more toxic than the crude venom (according to its fly larvae paralysing activity), is composed of 67 amino acids including six half cystines, and has an estimated molecular weight of 7532 and a p I value of 8.20. It causes an immediate contraction paralysis of fly larvae and a quick excitatory ‘knock-down’ effect on locusts. Insect toxin II (IT-II) is about 36-times more toxic than the crude venom according to the paralytic potency in fly larvae. It is composed of 69 amino acids including six half cystines and has an estimated molecular weight of 7894, a unique amino acid composition and a p I value of 8.30. IT-II causes a flaccid paralysis of fly larvae and a slow progressive paralysis and eventually death of locusts. In in vitro current clamp experiments, IT-I induces repetitive activities, and IT-II, a block of the evoked action potentials. These two opposite effects may be explained by their different effects on sodium permeabilities. Both toxins increase the sodium resting permeability (IT-II markedly more) resulting in a progressive depolarization of the axonal membrane. Concerning the activable sodium permeability, both toxins slightly slow the sodium transient inward current turning off. The peak sodium current is increased by IT-I and decreased byIT-II. This essential difference may, at least partially, account for the contrasting symptoms they induce in the whole insect.

Proteins in scorpion venoms toxic to mammals and insects

Abstract The crude venoms of the scorpions Androctonus aeneas aeneas , Androctonus amoreuxi , Androctonus mauretanicus mauretanicus , Buthus occitanus pairs , Buthus occitanus tunetanus and Leiurus quinquestriatus were submitted to starch gel zone electrophoresis. The eluates of gel sections were assayed for mice lethality, contraction-paralysis of fly larvae and larvae lethality. It has been found that: (1) All the venoms contain discrete components showing a specific toxicity to mice and to fly larvae. (2) Three of the venoms contain more than one larvae contraction-paralysis component. (3) At pH 8·6, most of the mice toxic components have a cathodic mobility whereas the majority of larvae toxic components migrate towards the anode. (4) All the larvae contraction-paralysis components are strongly lethal to fly larvae and are inactivated by trypsin, thus demonstrating their protein nature. (5) The venoms of A. a. aeneas and B. o. tunetanus contain factions of fast anodic mobility which are lethal to fly larvae but lack the larvae contraction-paralysis and the mice lethal activities. It is concluded that the presence of a variety of toxic proteins specifically active on different animals is a general property of scorpion venoms.

Functional expression of an alpha anti-insect scorpion neurotoxin in insect cells and lepidopterous larvae

The Leiurus quinquestriatus hebraeus alpha anti‐insect toxin (LqhαIT) cDNA was engineered into the Autographa californica Nuclear Polyhedrosis Virus (AcNPV) genome. Insect cells infected with the recombinant virus secreted a functional LqhαIT polypeptide. Spodoptera littoralis and Heliothis armigera larvae injected with recombinant budded virus, showed typical intoxication symptoms. This recombinant virus showed enhanced insecticidal potency against H. armigera larvae compared with wild type AcNPV. The present expression system will facilitate: (1) the future elucidation of structural elements involved in its prominent anti‐insect toxicity; and (2) the future design of genetically modified alpha toxins with improved anti‐insect selectivity.

Binding of an α scorpion toxin to insect sodium channels is not dependent on membrane potential

The insect‐specific LqhαIT toxin resembles α scorpion toxins affecting mammals by its amino acid sequence and effects on sodium conductance. The present study reveals that LqhαIT does not bind to rat brain membranes and possesses in locust neuronal membranes a single class of high affinity (K d = 1.06 ± 0.15 nM) and low capacity (B max = 0.7 ± 0.19 protein) binding sites. The latter are: (1) distinct from binding sites of other sodium channel neurotoxins; (2) inhibited by sea anemone toxin II; (3) cooperatively interacting with veratridine; (4) not dependent on membrane potential, in contrast to the binding sites of α toxins in vertebrate systems. These data suggest the occurrence of (a) conformational‐structural differences between insect and mammal sodium channels and (b) the animal group specificity and pharmacological importance of the α scorpion toxins.

A factor toxic to crustacean in the venom of the scorpion Androctonus australis hector

Abstract Pure neurotoxins separated from the venom of the scorpion Androctonus australis Hector and highly toxic to mammals are inactive when tested on several arthropods. The fly larvae toxin originating from the same venom demonstrates a strong toxicity to insects but is completely inactive when applied to an arachnid or a crustacean. Recycling gel filtration, on Sephadex G-50, of the crude venom allows the isolation of a product exhibiting a high paralyzing and lethal activity to a crustacean. The toxic activity is destroyed by trypsin digestion. It is concluded that, in addition to the toxins active in mammals and insects, the venom of A. australis contains another discrete protein specifically active on a crustacean.

A new toxic protein in the venom of the scorpion Androctonus australis Hector

Abstract By use of starch gel zone electrophoresis and Sephadex G-50 gel filtration a toxic protein with contraction-paralysis as well as lethal activity to fly larvae has been isolated from the venom of the scorpion Androctonus australis Hector . This larva-toxic protein is different from the mice-lethal neurotoxins of the same venom. The significance of this finding is discussed.