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Structure-function relationships of tetanus toxin
144
8th European Symposium-Abstracts
Addition of CIII or EQII to one side of a planar lipid membrane (HLM) increase the conductivity of the film in discrete steps of defined amplitude. High pH and sphingomyelin promote the interaction even in this system . We suggest that sea anemone cytolysins increase membrane permeability by forming discrete ion channels in it . REFERENCES Bextatn:n~te, A. W. and Rvnv, B. (1988) Biochim. Biophys. Acta 884, 12~141 . MENE3rRINA, G. (1988) FEBS Lett. 232, 217-220. Effects of myotoxin a on cultured muscle cells. ALLAN L. H®ea, H~~a A. Mrrnctnsox and C~r~ter.rs H. 7ror .tcowstn (Department of Chemistry, Arizona State University, Tempe, AZ, U.S .A.) . MYOTOXIN a is a small basic protein, isolated from the venom of Crotalus viridis viridis that causes extensive damage to muscle tissue when injected . We have used cultured myoblasts and the myotubes that result from cell fusion to study the effects of purified myotoxin a and some chemically modified forms of the protein . Standard cell culture techniques were used to establish and maintain clonal cell lines and primary cultures derived from neonatal rat skeletal muscle. Toxins were added to the cultures at concentrations which ranged from 0.0~ 1 .0 pM under well-defined culture conditions . The presence of myotoxin did not appear to affect the fusion process of primary myoblast cultures . Similar experiments with some clonal cell lines, however, led to the formation of very large myotubes compared to the untreated control cultures. Myotubes derived from primary cell cultures twitched spontaneously, but the twitching stopped when the normal growth medium was replaced with a defined serum-free medium . Addition of myotoxin a to the myotubes in the serum-free condition caused the twitching of the myotubes to resume . Crotamine, a closely related myotoxin from C. d. terriftcrrs venom, gave comparable results. Products derived from reductive alkylation, nitration and alkylation of myotoxin a were used in analogous experiments and were nearly devoid of activity when tested with the serum-free cell culture system . The results demonstrate the utility of muscle cell cultures in studies of myotoxin a and related compounds. Supported in part by National Institutes of Health; # 5 RO1 GM34925. Structure-junction relationships of tetanus toxin. HERNARD Brzztrtr, (Pasteur Institute, Paris, France). TsrAxus toxin has been extensively studied over the last two decades. The physicochemical and toxicological characteristics, as well as the secondary structure of the toxin purified to homogeneity have been specified. The toxin molecule has been sequenced. The binding characteristics have been determined and the axoplasmic and transynaptic movement of the toxin demonstrated. However, its mode of action is still to be clarified. In this lecture we shall report on our own contribution to the studies on tetanus toxin in relation to the structur~function relationships: mechanism of tetanus toxin detoxification by formaldehyde action and its implication for vaccine preparation; involvement of particular amino acid residues in toxin action; toxicity and immunological reactivity and significance for the preparation of therapeutic antisera; protease cleavage of the toxin molecule, purification from the cleaved molecule of a fragment ; H~, involved in binding and transport of the toxin molecule to the CNS and of another fragment, L-H,, responsible for the paralytic (botulinum toxin-like effect); use of the HZ fragment for targeting an enzyme or a drug to the CNS; application to the treatment of a grafted brain tumour ; use of HZ as a tool for investigating the bases of "neurotropism" ; its interaction with the fate of a neurotropic virus inoculated by i.v. or i.m. routes; investigation of the pathogenic action of fragment L-H, in its native state, in situ in the toxin molecule; interaction of the toxin molecule and the L-H,-derived fragment with guanylate cyclase. Activity of ~yotoxin P, from the venom of Naja nigricollis after various chemical modt~cations. GAnt HORKOW, AnrxA C~rArur-MA1YA3 and MrcxAaL t)vAnrA, (Department of Zoology, Tel Aviv University, Israel 69978). Ttm vsxoes of Naja nigricollis includes four cytotoxic factors (P 3P~, Ctwta-MA~rvAS and twAnu, 1989). The myotoxin P, was isolated in three steps: (l) reduction by 1% 2-mercaptcethanol; (2) gel filtration on Sephadex G-50; (3) ion-exchange chromatography on CM-Sepharose. The cytotoxic activity was tested on melanoma cells B16F10 and on WEHI-3B leukemia cells. The purified myotoxin P, was modified by various reagents reacting with different residues of the protein. The N-terminal and the C-terminal groups of the polypeptide were modified by N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or cystamine respectively; these reagents include 5-S bonds. Both modified polypeptides retained about 90% of their original cytotoxic activity . Moreover, reduction of the S~ bonds of the bound reagents in mild conditions exposed the SH groups on the reagents without affecting the cytotoxicity of the polypeptide . However, at extreme reducing conditions, the integral S-S bonds of the ~yotoxin were also reduced, leading to a total loss of the cytotoxic activity . Similarly, other modifications such as succination, maleylation, reductive alkylation, carbamylation, reaction with 1,2cyclohexanedione or complete iodination of the toxin also abolished the cytotoxic activity, but did not affect its
8th European Symposium-Abstracts
Addition of CIII or EQII to one side of a planar lipid membrane (HLM) increase the conductivity of the film in discrete steps of defined amplitude. High pH and sphingomyelin promote the interaction even in this system . We suggest that sea anemone cytolysins increase membrane permeability by forming discrete ion channels in it . REFERENCES Bextatn:n~te, A. W. and Rvnv, B. (1988) Biochim. Biophys. Acta 884, 12~141 . MENE3rRINA, G. (1988) FEBS Lett. 232, 217-220. Effects of myotoxin a on cultured muscle cells. ALLAN L. H®ea, H~~a A. Mrrnctnsox and C~r~ter.rs H. 7ror .tcowstn (Department of Chemistry, Arizona State University, Tempe, AZ, U.S .A.) . MYOTOXIN a is a small basic protein, isolated from the venom of Crotalus viridis viridis that causes extensive damage to muscle tissue when injected . We have used cultured myoblasts and the myotubes that result from cell fusion to study the effects of purified myotoxin a and some chemically modified forms of the protein . Standard cell culture techniques were used to establish and maintain clonal cell lines and primary cultures derived from neonatal rat skeletal muscle. Toxins were added to the cultures at concentrations which ranged from 0.0~ 1 .0 pM under well-defined culture conditions . The presence of myotoxin did not appear to affect the fusion process of primary myoblast cultures . Similar experiments with some clonal cell lines, however, led to the formation of very large myotubes compared to the untreated control cultures. Myotubes derived from primary cell cultures twitched spontaneously, but the twitching stopped when the normal growth medium was replaced with a defined serum-free medium . Addition of myotoxin a to the myotubes in the serum-free condition caused the twitching of the myotubes to resume . Crotamine, a closely related myotoxin from C. d. terriftcrrs venom, gave comparable results. Products derived from reductive alkylation, nitration and alkylation of myotoxin a were used in analogous experiments and were nearly devoid of activity when tested with the serum-free cell culture system . The results demonstrate the utility of muscle cell cultures in studies of myotoxin a and related compounds. Supported in part by National Institutes of Health; # 5 RO1 GM34925. Structure-junction relationships of tetanus toxin. HERNARD Brzztrtr, (Pasteur Institute, Paris, France). TsrAxus toxin has been extensively studied over the last two decades. The physicochemical and toxicological characteristics, as well as the secondary structure of the toxin purified to homogeneity have been specified. The toxin molecule has been sequenced. The binding characteristics have been determined and the axoplasmic and transynaptic movement of the toxin demonstrated. However, its mode of action is still to be clarified. In this lecture we shall report on our own contribution to the studies on tetanus toxin in relation to the structur~function relationships: mechanism of tetanus toxin detoxification by formaldehyde action and its implication for vaccine preparation; involvement of particular amino acid residues in toxin action; toxicity and immunological reactivity and significance for the preparation of therapeutic antisera; protease cleavage of the toxin molecule, purification from the cleaved molecule of a fragment ; H~, involved in binding and transport of the toxin molecule to the CNS and of another fragment, L-H,, responsible for the paralytic (botulinum toxin-like effect); use of the HZ fragment for targeting an enzyme or a drug to the CNS; application to the treatment of a grafted brain tumour ; use of HZ as a tool for investigating the bases of "neurotropism" ; its interaction with the fate of a neurotropic virus inoculated by i.v. or i.m. routes; investigation of the pathogenic action of fragment L-H, in its native state, in situ in the toxin molecule; interaction of the toxin molecule and the L-H,-derived fragment with guanylate cyclase. Activity of ~yotoxin P, from the venom of Naja nigricollis after various chemical modt~cations. GAnt HORKOW, AnrxA C~rArur-MA1YA3 and MrcxAaL t)vAnrA, (Department of Zoology, Tel Aviv University, Israel 69978). Ttm vsxoes of Naja nigricollis includes four cytotoxic factors (P 3P~, Ctwta-MA~rvAS and twAnu, 1989). The myotoxin P, was isolated in three steps: (l) reduction by 1% 2-mercaptcethanol; (2) gel filtration on Sephadex G-50; (3) ion-exchange chromatography on CM-Sepharose. The cytotoxic activity was tested on melanoma cells B16F10 and on WEHI-3B leukemia cells. The purified myotoxin P, was modified by various reagents reacting with different residues of the protein. The N-terminal and the C-terminal groups of the polypeptide were modified by N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or cystamine respectively; these reagents include 5-S bonds. Both modified polypeptides retained about 90% of their original cytotoxic activity . Moreover, reduction of the S~ bonds of the bound reagents in mild conditions exposed the SH groups on the reagents without affecting the cytotoxicity of the polypeptide . However, at extreme reducing conditions, the integral S-S bonds of the ~yotoxin were also reduced, leading to a total loss of the cytotoxic activity . Similarly, other modifications such as succination, maleylation, reductive alkylation, carbamylation, reaction with 1,2cyclohexanedione or complete iodination of the toxin also abolished the cytotoxic activity, but did not affect its