Serge Lissitzky

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.

Scorpion neurotoxins: A family of homologous proteins.

Eleven neurotoxins have been purified from the venoms of three scorpion sub-species, Androctonus australis Hector, Buthus occitanus tunetanus and Leiurus quinquestriatus quinquestriatus [ 1 ] . These proteins all consist of a single polypeptide chain of 57 to 66 amino acid residues cross-linked by four disulfide bridges. The complete amino acid sequence of toxins I and I’ of A. australis Hector has been recently determined [2,3]. In this communication we report the sequence of the first 22 to 26 amino acid residues from the N-terminal end of six additional neurotoxins. It is shown that scorpion neurotoxins form a new set of homologous proteins. Furthermore, this set can be divided in three subgroups when additional amino acid sequence homologies and specific toxities are taken into account.

Radioiodination of scorpion and snake toxins.

Abstract Several scorpion and snake toxins were radioiodinated using the lactoperoxydase method of [125I]iodide oxidation. Two techniques of labeling were set up: (i) Using carrier-free Na125I and 5 μg of toxin, about one iodine atom was incorporated per mole of protein without loss of toxicity. Specific radioactivities about 2,000 Ci/mmol (280 μCi/μg) were obtained. The modified toxin, purified by immunoprecipitation with an antiserum prepared against the native toxin, was obtained in a short time (4 hr), with a good yield (50 to 80%), and in a small volume (1 ml). (ii) Using Na127I traced with Na 125I and larger amounts (200 μg) of toxin, more than one iodine atom was incorporated per mole of protein without loss of activity. Lower specific radioactivities (1 to 1.5 Ci/mmol) were obtained. The iodinated toxins were purified by gel filtration of the radioiodination mixtures on a column made of two layers of Sephadex (G-15 and G-50). The modified proteins were extensively analyzed by paper electrophoresis and polyacrylamide gel electrophoresis. Their content of monoiodotyrosine and diiodotyrosine was estimated and, in the case of toxin I of Androctonus australis Hector, it was possible to follow the iodination rate of its three tyrosine residues by automatic Edman degradation. The mode of purification of the iodinated scorpion toxins affects their behavior on molecular sieving on Sephadex G-50 and on electrophoresis on polyacrylamide gel. The results are discussed.

Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells

Abstract The protein neurotoxin II from the venom of the scorpion Androctonus australis Hector was labeled with 125I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (KD = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na+ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone Anemona sulcata also affects the closing of the action potential Na+ ionophore in nerve axons. The unlabelled sea anemone toxin modifies 125I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent KD for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both Androctonus scorpion toxin II and Anemona sea anemone toxin II interact competitively with a regulatory component of the action potential Na+ channel.

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.

Thyroglobulin iodoamino acids estimation after digestion with pronase and leucylaminopeptidase

A method for the quantitative estimation of thyroglobulin iodoamino acid residues is described. It combines (1) digestion of thyroglobulin (or iodinated proteins) with pronase (10% by weight for 48 hr) and leucylaminopeptidase (20% by weight for 24 hr), (2) separation of the free iodoamino acids liberated by chromatography on a cation-exchange resin column (Dowex 50-X4) and their quantitative estimation by the sodium arsenite/ceric sulfate reaction operated automatically according to a modification of the procedure of Block and Mandl. The complete release of iodotyrosines and iodothyronines is obtained in the case of man and horse thyroglobulins. For other thyroglobulins (rat, sheep, ox, dog) undigested iodinated material represents between 3 and 6% of total iodine. Deiodination is low and never exceeds around 3% of total protein iodine. The procedure described in this paper definitely improves procedures that are already known.

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.

Thyroid-stimulating hormone binding to cultured thyroid cells

The suggestion that thyroid stimulating hormone (TSH) interacts with its target tissue at a superficial cell site was made by Pastan et al. [I] on the basis of experiments showing the persistent effect of TSH on [ l-14C]glucose oxidation in thyroid slices exposed to the hormone and washed thoroughly in hormone-free medium. Additional support to this idea was given by Yamashita and Field [2] and Wolff and Jones [3] who showed that plasma membranes purified from thyroid gland contained a TSH-sensitive adenylcyclase. Previous investigations demonstrated that TSH induced the reorganization into follicles of cultured isolated thyroid cells (Fayet and Tixier [4], Fayet et al. [5] ), via the adenylcyclase-cyclic AMP system (Fayet and Lissitzky [6], Lissitzky et al. [7]) whereas monolayer cultures were obtained in its absence. TSHor dibutyryl cyclic AMP-stimulated cells showed the organizational [8] and the specific metabolic properties of the gland follicular cells [7]. This communication reports the direct measurement of specific TSH-receptor interaction in this system and some of the properties of the binding sites. 2. Materials and methods