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2D NMR spectroscopy in toxin research
9th World Congress
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secretion and/or ionic channel, etc. The model (OLSNES et al., 1974) can be extended to several toxic proteins including those derived from bacterial origin such as the botulinum, tetanus, cholera and diphtheria toxins. Some other toxins from animal origin are also candidates for the haptomer--effectomer model. Supported by FINEP, CNPq, CEPG-UFRJ and IFS (Sweden). REFERENCES CARLINI et al. (1988) Phytochemistry 27, 25. OLSNES et al. (1974) Nature 249, 627.
Pathogenesis of myonecrosis induced by a myotoxin isolated from the venom of Bothrops nummifier (Mano de piedra). Josf~ MARIA GUTn~RREZ, FERNANDO CHAVES, JOSI~ A. GENI~, BRUNO LOMONTE and ZIANE CAMACHO (Instituto CIodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, San Josr, Costa Rica). A MYOTOXINwas isolated from the venom of Bothrops nummifer. It has a mol.wt of 13,000 and a high isoelectric point. The toxin lacks phospholipase A2 activity, although its amino acid composition has similarities with those of phospholipases A 2. Upon i.m. injection in mice the toxin induces prominent myonecrosis. Histological changes were followed, and affected cells showed the presence of 'delta' lesions, whereas others had a clumped morphology. The toxin induced an increase in calcium levels of skeletal muscle, and a decrease in muscle creatine and creatine kinase contents. The toxin disrupted the integrity of liposomes made of phospholipids extracted from skeletal muscle, cardiac muscle and erythrocytes. Myotoxin had an amphipatic behaviour when analyzed by charge-shift electrophoresis. On the basis of the evidence collected, it is proposed that B. nummifer myotoxin induces muscle damage by first affecting the integrity of skeletal muscle plasma membrane, perhaps due to a toxin-induced disorganization of membrane phospholipids.
Palytoxin acts through Na +, K+-ATPase. ERNST HABERMANN,1 HEINER BOTTINGER,2 GURSHARAN S. CHHATWAL2 and MARIE E. DAUZENROTH1 (~Departments of Pharmacology and 2Immunology and Microbiology, Justus-Liebig-University, D-6300 Giessen, F.R.G.). PALYTOXIN increases the permeability of many cells to small ions. On human erythrocytes, the resulting complete release of K ÷ is prevented by ouabain. The glycoside concomitantly inhibits binding of 12SI-palytoxin, and palytoxin inhibits binding of [3H]-ouabain. These and other findings suggest Na ÷, K+-ATPase as the target of palytoxin. We have now looked for additional receptors. (l) Dog erythrocytes are nearly devoid of Na +, K+-ATPase. Accordingly, here the efficacy of palytoxin is very low, however ouabain-sensitive. Raterythrocytes are sensitive to palytoxin. Their K + release is insensitive to ouabain in the presence of but sensitive in the absence of Ca 2+. (2) [3H]-Dopamine release from rat pheochromocytoma cells can be triggered by palytoxin. Since Ca 2 + has to be added to allow release, inhibition by ouabain is absent. (3) Human HeLa cells release K + when exposed to palytoxin. Ouabain prevents the palytoxin effect. All data can be explained by the specific interaction of palytoxin with Na ÷, K+-ATPase, and the atypical behaviour of rat ATPase in the presence of Ca 2+. Evidences for an additional receptor have not been found. REFERENCES BISTTINGER, H., BERESS, L. and HABERMANN,E. (1986) Biochim. biophys. Acta 861, 165. CHHATWAL, G. S., HESSLER, H.-J. and HABERMANN, E. (1983) Naunyn-Schmiedeberg's Arch. Pharmac. 323, 261.
2D NMR spectroscopy in toxin research. GERHARD G. HABERMEHL (Department of Chemistry, Veterinary University, D-3000 Hannover 1, F.R.G.). AMONG the physical methods used in structure elucidation of complicated molecules, e.g. toxins, N M R spectroscopy has gained more and more importance, especially as 2-dimensional 1H, ~H- or
n,13C-measurements. This method is described using examples of different toxins from plants and animals. Among plant toxins the structure determination of macrocyclic trichothecenes as well as macrocyclic alkaloids is discussed. From animals low molecular peptides are used as examples for structure elucidation. Isolation and purification of such substances from snake venoms (Bothrops jararacussu, B. moojeni and B. atrox) are also reported. These substances are of special interest as they have hardly been investigated so far. They do not belong to the groups of toxins or enzymes but they possess other biologic activities, e.g. as potentiators.
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secretion and/or ionic channel, etc. The model (OLSNES et al., 1974) can be extended to several toxic proteins including those derived from bacterial origin such as the botulinum, tetanus, cholera and diphtheria toxins. Some other toxins from animal origin are also candidates for the haptomer--effectomer model. Supported by FINEP, CNPq, CEPG-UFRJ and IFS (Sweden). REFERENCES CARLINI et al. (1988) Phytochemistry 27, 25. OLSNES et al. (1974) Nature 249, 627.
Pathogenesis of myonecrosis induced by a myotoxin isolated from the venom of Bothrops nummifier (Mano de piedra). Josf~ MARIA GUTn~RREZ, FERNANDO CHAVES, JOSI~ A. GENI~, BRUNO LOMONTE and ZIANE CAMACHO (Instituto CIodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, San Josr, Costa Rica). A MYOTOXINwas isolated from the venom of Bothrops nummifer. It has a mol.wt of 13,000 and a high isoelectric point. The toxin lacks phospholipase A2 activity, although its amino acid composition has similarities with those of phospholipases A 2. Upon i.m. injection in mice the toxin induces prominent myonecrosis. Histological changes were followed, and affected cells showed the presence of 'delta' lesions, whereas others had a clumped morphology. The toxin induced an increase in calcium levels of skeletal muscle, and a decrease in muscle creatine and creatine kinase contents. The toxin disrupted the integrity of liposomes made of phospholipids extracted from skeletal muscle, cardiac muscle and erythrocytes. Myotoxin had an amphipatic behaviour when analyzed by charge-shift electrophoresis. On the basis of the evidence collected, it is proposed that B. nummifer myotoxin induces muscle damage by first affecting the integrity of skeletal muscle plasma membrane, perhaps due to a toxin-induced disorganization of membrane phospholipids.
Palytoxin acts through Na +, K+-ATPase. ERNST HABERMANN,1 HEINER BOTTINGER,2 GURSHARAN S. CHHATWAL2 and MARIE E. DAUZENROTH1 (~Departments of Pharmacology and 2Immunology and Microbiology, Justus-Liebig-University, D-6300 Giessen, F.R.G.). PALYTOXIN increases the permeability of many cells to small ions. On human erythrocytes, the resulting complete release of K ÷ is prevented by ouabain. The glycoside concomitantly inhibits binding of 12SI-palytoxin, and palytoxin inhibits binding of [3H]-ouabain. These and other findings suggest Na ÷, K+-ATPase as the target of palytoxin. We have now looked for additional receptors. (l) Dog erythrocytes are nearly devoid of Na +, K+-ATPase. Accordingly, here the efficacy of palytoxin is very low, however ouabain-sensitive. Raterythrocytes are sensitive to palytoxin. Their K + release is insensitive to ouabain in the presence of but sensitive in the absence of Ca 2+. (2) [3H]-Dopamine release from rat pheochromocytoma cells can be triggered by palytoxin. Since Ca 2 + has to be added to allow release, inhibition by ouabain is absent. (3) Human HeLa cells release K + when exposed to palytoxin. Ouabain prevents the palytoxin effect. All data can be explained by the specific interaction of palytoxin with Na ÷, K+-ATPase, and the atypical behaviour of rat ATPase in the presence of Ca 2+. Evidences for an additional receptor have not been found. REFERENCES BISTTINGER, H., BERESS, L. and HABERMANN,E. (1986) Biochim. biophys. Acta 861, 165. CHHATWAL, G. S., HESSLER, H.-J. and HABERMANN, E. (1983) Naunyn-Schmiedeberg's Arch. Pharmac. 323, 261.
2D NMR spectroscopy in toxin research. GERHARD G. HABERMEHL (Department of Chemistry, Veterinary University, D-3000 Hannover 1, F.R.G.). AMONG the physical methods used in structure elucidation of complicated molecules, e.g. toxins, N M R spectroscopy has gained more and more importance, especially as 2-dimensional 1H, ~H- or
n,13C-measurements. This method is described using examples of different toxins from plants and animals. Among plant toxins the structure determination of macrocyclic trichothecenes as well as macrocyclic alkaloids is discussed. From animals low molecular peptides are used as examples for structure elucidation. Isolation and purification of such substances from snake venoms (Bothrops jararacussu, B. moojeni and B. atrox) are also reported. These substances are of special interest as they have hardly been investigated so far. They do not belong to the groups of toxins or enzymes but they possess other biologic activities, e.g. as potentiators.