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Antigenically active, two complementary polypeptide fragments of tetanus neurotoxin
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4th International Symposium on Animal, Plant and Microbial Toxins
(b) Trichogaster fasciatua. The chief lesion was degeneration of the cytoplasm of the cells in the form of vacuoles . In severe cases the centre of the cells appeared empty. At a few places localized necrosis was much marked. Hypertrophy was not noticed . MATHUx, D . S ., Toxicon 8,141, 1972 . MATSUDA, M. and YoNEDA, M., Research Institute for Microbial Diseases, Osaka University, Yamada-
kami, Suita, Osaka 565, Japan.
ACTIVE, TWO COMPLEMENTARY POLYPEPTIDE FRAGMENTS OF TETANUS NEUROTOXIN In our laboratory, attempts have been made to obtain a clue for the elucidation of structure-function relationship of tetanus neurotoxin . Analyses using polyacrylamide gel electrophoresis revealed that the toxin as purified from culture filtrates (`extracellular' toxin, mol. wt . 160,000) could be dissociated into two polypeptide chains of molecular weight 53,000 (Fragment a) and 107,000 (Fragment ß) by treatment with dithiothreitol (DTT) and sodium dodecyl sulfate (SDS) or with DTT and urea. Urea-, but not SDS-, treatment of the thiol reduced toxin resulted in reversible dissociation of the toxin into Fragments a and ß . Thus removal of DTI' and urea by dialysis restored the toxicity of the DTT-urea-treated toxin with concomitant reassociation of the fragments . Isolation and purification of the two polypeptide fragments from the DTT-urea-treated toxin were then carried out by procedures including electrophoresic separation on 2M urea-polyacrylamide gel and subsequent electrophoresic elution from the macerated gel slices containing the corresponding protein band . Fragments a and ß thus purified were found to be antigenically active . Immunodiffusion analyses using horse antitoxin showed that they are distinct antigenicafy but partially identical with the undissociated toxin in antigenicity. The antitoxin precipitating ability of both fragments was inactivated by heating at 60 ° C for 10 min . Neither fragments showed tetanospasmin activity. Investigation on detailed intmunological and chemical properties of the two fragments is now in progress. ANTIGENICALLY
Was, D.,1 LEE, C . Y., Cremt, Y . M .,' and IwANAoA, S.' (1) Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt a.M ., Germany, (2) Pharmacological Institute, College of Medicine, National Taiwan University, Tapei and (3) Institute for Protein Research, Osaka University, Suita, Osaka, Japan . CHEMICAL AND PHARMACOLOGICAL CHARACTERIZATION OF TOXIC POLYPEPTIDES FROM FOUR ELAPIDAE VENOMS Venom of the Indian krait Bungarus caeruleus, the Egyptian desert black snake Walterinnesia aegyptia and two Australian Elapidae snakes, the copperhead Denisonia superba and the black snake Pseudechis porphyriacus have been fractionated by column chromatography on CM-cellulose . The fractions which exhibited toxic symptoms in mice after subcutaneous injection were further purified by rechromatography on CM-Sephadex C-25 and gel filtration on Sephadex G-50. Their toxicity (LD, .) was determined and their effects on the neuromuscular transmission were investigated. Those fractions which showed homogeneity in disc-electrophoresis were characterized in their amino acid composition . Some structural properties will also be reported . MENDEtssoHN, H ., Tel-Aviv University, Department of Zoology, 155 Herzl Street, Tel-Aviv, Israel . ECOLOGY OF VENOMOUS SNAKES IN HUMAN SETTLEMENTS Abstract not received MENEz, A. and FttomAC)EoT, P ., Service de Biochimie, Centre d'Etudes Nucléaires de Saclay, France. SOME BIOLOGICAL PROPERTIES OF TWO TRITIATED NEUROTOXINS Snake neurotoxins are known to act at the neuromuscular junction by blocking the cholinergic receptor sites. The characterization of such sites required highly purified and labelled neurotoxins . We report the labelling of the a toxin of Naja n1grtcollis and of Erabutoxin b from Laticaudasemifasciata. The technique used consisted in the hydrogenolysis of an iodohistidine residue (nos. 4 and 26, respectively) of the peptide . The specific radioactivity obtained reached 27 Ci per mmole and 1 Ci per numole, respectively. The physiochemical identities between the tritiated and the parent compounds led us to compare the binding affinities for the receptor cholinergic sites, partially purified from Torpedo nrarmorata, according TOXICON1975 vor. 13
4th International Symposium on Animal, Plant and Microbial Toxins
(b) Trichogaster fasciatua. The chief lesion was degeneration of the cytoplasm of the cells in the form of vacuoles . In severe cases the centre of the cells appeared empty. At a few places localized necrosis was much marked. Hypertrophy was not noticed . MATHUx, D . S ., Toxicon 8,141, 1972 . MATSUDA, M. and YoNEDA, M., Research Institute for Microbial Diseases, Osaka University, Yamada-
kami, Suita, Osaka 565, Japan.
ACTIVE, TWO COMPLEMENTARY POLYPEPTIDE FRAGMENTS OF TETANUS NEUROTOXIN In our laboratory, attempts have been made to obtain a clue for the elucidation of structure-function relationship of tetanus neurotoxin . Analyses using polyacrylamide gel electrophoresis revealed that the toxin as purified from culture filtrates (`extracellular' toxin, mol. wt . 160,000) could be dissociated into two polypeptide chains of molecular weight 53,000 (Fragment a) and 107,000 (Fragment ß) by treatment with dithiothreitol (DTT) and sodium dodecyl sulfate (SDS) or with DTT and urea. Urea-, but not SDS-, treatment of the thiol reduced toxin resulted in reversible dissociation of the toxin into Fragments a and ß . Thus removal of DTI' and urea by dialysis restored the toxicity of the DTT-urea-treated toxin with concomitant reassociation of the fragments . Isolation and purification of the two polypeptide fragments from the DTT-urea-treated toxin were then carried out by procedures including electrophoresic separation on 2M urea-polyacrylamide gel and subsequent electrophoresic elution from the macerated gel slices containing the corresponding protein band . Fragments a and ß thus purified were found to be antigenically active . Immunodiffusion analyses using horse antitoxin showed that they are distinct antigenicafy but partially identical with the undissociated toxin in antigenicity. The antitoxin precipitating ability of both fragments was inactivated by heating at 60 ° C for 10 min . Neither fragments showed tetanospasmin activity. Investigation on detailed intmunological and chemical properties of the two fragments is now in progress. ANTIGENICALLY
Was, D.,1 LEE, C . Y., Cremt, Y . M .,' and IwANAoA, S.' (1) Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt a.M ., Germany, (2) Pharmacological Institute, College of Medicine, National Taiwan University, Tapei and (3) Institute for Protein Research, Osaka University, Suita, Osaka, Japan . CHEMICAL AND PHARMACOLOGICAL CHARACTERIZATION OF TOXIC POLYPEPTIDES FROM FOUR ELAPIDAE VENOMS Venom of the Indian krait Bungarus caeruleus, the Egyptian desert black snake Walterinnesia aegyptia and two Australian Elapidae snakes, the copperhead Denisonia superba and the black snake Pseudechis porphyriacus have been fractionated by column chromatography on CM-cellulose . The fractions which exhibited toxic symptoms in mice after subcutaneous injection were further purified by rechromatography on CM-Sephadex C-25 and gel filtration on Sephadex G-50. Their toxicity (LD, .) was determined and their effects on the neuromuscular transmission were investigated. Those fractions which showed homogeneity in disc-electrophoresis were characterized in their amino acid composition . Some structural properties will also be reported . MENDEtssoHN, H ., Tel-Aviv University, Department of Zoology, 155 Herzl Street, Tel-Aviv, Israel . ECOLOGY OF VENOMOUS SNAKES IN HUMAN SETTLEMENTS Abstract not received MENEz, A. and FttomAC)EoT, P ., Service de Biochimie, Centre d'Etudes Nucléaires de Saclay, France. SOME BIOLOGICAL PROPERTIES OF TWO TRITIATED NEUROTOXINS Snake neurotoxins are known to act at the neuromuscular junction by blocking the cholinergic receptor sites. The characterization of such sites required highly purified and labelled neurotoxins . We report the labelling of the a toxin of Naja n1grtcollis and of Erabutoxin b from Laticaudasemifasciata. The technique used consisted in the hydrogenolysis of an iodohistidine residue (nos. 4 and 26, respectively) of the peptide . The specific radioactivity obtained reached 27 Ci per mmole and 1 Ci per numole, respectively. The physiochemical identities between the tritiated and the parent compounds led us to compare the binding affinities for the receptor cholinergic sites, partially purified from Torpedo nrarmorata, according TOXICON1975 vor. 13