Herve Rochat

Immunochemistry of scorpion α-toxins: Antigenic homologies checked with radioimmunoassays (RIA)

Three toxins, i.e. toxins II and III of Androctonus australis Hector and toxin I of Buthus occitanus tunetanus, were labeled with 125I. High specific radioactivities were obtained (490–1100 Ci/mmole) that allowed the setting up to three radioimmunoassays. We were able to quantify the amount of toxin in 0.1 ml of 10−9–10−10 M solutions and to test antigenic homologies between toxins belonging to different structural groups. Three possibilities exist: (1) no cross-reactivity when sequence difference is greater than 25%; (2) full-cross-reactivity when this difference is lower than 25%; (3) partial cross-reactivity, which is interpreted as a loss of some common antigenic sites.

Purification of animal neurotoxins: isolation and characterization of three neurotoxins from the venom of Naja nigricollis mossambica peters.

This work is part of a general study on snake neurotoxins. In previous papers [1,2], we had shown that the application to snake venoms of the general method of purification which was developed to isolate toxins from scorpion venoms [3-7] was very efficient. Four neurotoxins from two different sources ofNaja haje venom [1] and two neurotoxins fromNaja nigricollis venom [2] were purified when only one toxin had been previously isolated in a pure form from each of these venoms by others [8,9]. N-terminal sequences determination using automatic Edman degradation, was found to be the best method for rapid characterization of newly purified toxins [2]. We report, in this paper, on the application of these methods to the venom of Naja nigricollis mossambica Peters. The partial sequences of the neurotoxins show that these are more closely related to cobrotoxin (a toxin isolated from the venom of the formosan Elapidae Naja naja atra) than the toxins I and II isolated from the venom ofNaja nigricollis living in Ethiopia [2]. The taxonomical importance of these results is discussed.

Use of parvalbumin as a protecting protein in the sequenator: An easy and efficient way for sequencing small amounts of peptides

Since the description of the first automated apparatus for degradation of proteins by the phenylisothiocyanate method [l] it became obvious that the procedures worked out by Edman and Begg and later by Hermodson et al. [2] were not directly applicable to small and hydrophobic peptides. It has been shown that this was due to losses of the residual peptide during the extraction steps with organic solvents [ 1,3-61. Several alternative approaches have been developed to overcome the problem. One of these is to render the peptide suitable for the usual procedures by coupling polar groups either to lysine [4 ] and S-@-aminoethyl) cysteine residues [7] or to the C-terminal amino acid [.5,8]. A second approach is to adapt the procedure to the peptide either by decreasing the concentration of quadrol [6,8,9] or by using a volatile buffer [8] which, in both cases, limits the number and the volumes of organic solvents used for extraction. A third possibility is to attach the peptide to an insoluble resin and perform the degradation in an heterogeneous phase [lo] with the help of a specially designed apparatus [ 111. We wish to report the results we obtained in sequencing unmodified peptides in very small amounts (down to 20 nmol) in an Edman and Begg type sequenator using dimethylbenzylamine as coupling buffer but in the presence of a naturally aNHz-blocked protein (hake major parvalbumin) that acts as a protecting film. The technique proved also to be very efficient for the degradation of peptides attached to polystyrene resins: it prevents the loss

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells

Scorpion toxins, the basic miniproteins of scorpion venom, stimulated the passive uptake of Na+ and Ca2+ in chick embryo heart cells. Half-maximum stimulation was obtained for 20-30 nM Na+ and 40-50 nM Ca2+. Scorpion toxin-activated Na+ and Ca2+ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na+ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nM) for both Na+ and Ca2+. Scorpion toxin-stimulated Ca2+ uptake was dependent on extracellular Na+ concentration and was not inhibited by Ca2+ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca2+ transport, which is coupled to passive Na+ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin - sensitive fast channels.

Evidence for the existence of two isocolipases in horse pancreas

Abstract The N-terminal amino acid sequences of two forms of colipase isolated from horse pancreas have been compared. Four sequence differences were found in the first 51 amino acids. This lead us to conclude that there are two distinct colipases in the horse pancreas.

A sensitive radioimmunoassay of a scorpion neurotoxin Toxin I of Androctonus australis Hector

Scorpion neuro tox ins fo rm a family o f homologous proteins which are basic and have approx , rnol. wt 7000 [ l ] . They consist o f a s in#e pept ide chain cross!inked b y four disulfide bridges [2i. The complete amino acid sequences oI some o f t h e m [3,4L as well as the N-term!ha ~. c f o thers , have been determined: their compar i son has led to a classification into four groups [5 i . They have been shown to affect the conduc t ion o f ions th rough mere brane channels [ 6 I 0 ] , and are thus good tools for the s tudy o f t h e s e s t r u c t u r e s o n t h e m o l e c u l a r l eve l . T o x i n s I a n d H o f A n d r o c t o n u s ausgralis Hec tor have been ~2s[_ labeled: specific radioactivRies up to 2000 C i /mmol have been obta ined [11 ,12] . We repor t here the set t ing up o f a radioh-amunoassay allowing a sensitive and specific de tec t ion o f toxin I o f Androc tonus australis Hector.

A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin AahI.

Abstract We created a construct encoding a peptide known to mimic the binding properties of biotin fused to the carboxyterminus of a scFv fragment that binds a scorpion toxin (AahI). This fusion protein was produced in the periplasm of bacteria and purified to homogeneity by singlestep affinity chromatography on streptavidinagarose with a yield close to 1 mg/l. DNA sequencing, dot blot and mass spectrometric analyses demonstrated the integrity of the soluble immunoconjugate. Fusion to the streptavidinbinding peptide did not affect the ability of the scFv to recognize its antigen with a high affinity (Kd = 2.3x 1010M). Similarly, the streptavidinbinding property was not impaired in the fusion protein. Thus, the immunoconjugate was bifunctional and had a low molecular mass of 28 kDa. This enabled us to develop rapid and sensitive immunoassays for the specific detection of the toxin AahI accurately to 0.6 ng/ml, opening up new perspectives for the diagnosis of envenomations.

The amino acid sequence of cytochrome c from Solanum tuberosum (potato)

The primary structure of potato (Sofanum tuberosum) cytochrome c has been determined using 1.5 pmole of protein. When compared to the sequence of tomato (Lycopersicum esculentum) cytochrome c which had been determined by R. Scogin et al. [l] , only five differences are observed in the positions 4, 29, 58,66 and 98. These two proteins belong to two genus of the same family (Solanaceue). The homology is strong with other higher plants cytochromes c yet described.

Cobra venom phospholipase highly toxic to arthropods—I. Purification and characterization

It has been found that the potent toxicity of Naja mossambica mossambica Cobra snake venom to arthropods is due substantially to a basic phospholipase A2. This main conclusion is supported by the following data: 1. (1) The isolation and purification of the active compound was achieved using Sephadex G50 gel filtration and Biorex 70 ion exchange chromatography. The resulting product possessed 65% of the total toxicity of the venom and a 13-fold higher specific toxicity. 2. (2) The homogeneity of the toxic factor was assessed by disc-electrophoresis, immunoelectrophoresis, analytical ultracentrifugation, isoelectrofocusing, amino acid analysis and C-terminal as well as N-terminal sequence determinations. 3. (3) The toxic factor (P3) is a single chain basic protein (pHi =9.63) made up from 118 amino acids (Mol. wt. 13,200) cross-linked by seven disulphide bridges and has a tendency to dimerize. 4. (4) The toxic factor possesses phospholipatic activity hydrolysing phospholipids to release fatty acids. Positional specificity shown toward the fatty acids of phosphatidylcholine indicates that it is an A2 type phospholipase (E C 3.1.1.4). Additional characteristics such as basic pH (8–10) and high temperature (50–70°C) optima and calcium dependent activity are all typical of the A2 group of phospholipases. The possible relations between the enzymatic and toxic properties of this protein are discussed.

Parvalbumins from coelacanth muscle II. Amino acid sequence of the two less acidic components

The primary structure of the two less acidic parvalbumins (pI = 5.44 and pI = 4.95) from coelacanth muscle (Latimeria chalumnae) has been determined. They differ only by the presence or absence of a N-terminal blocking group. By the use of the automatic degradation, 69 amino acids could be placed unambiguously in the N-terminal part and 24 amino acids following the single arginine 75. Tryptic peptides were used to establish the sequence and the position of the remaining residues. The two parvalbumins examined belong to the alpha-lineage, and the rate of their molecular evolution is comparable to that found in other vertebrates.