Botulinum neurotoxin, produced by the ubiquitous spore forming bacteria Clostridium botulinum, is the primary cause of botulism. The disease generally results from food poisoning. Infant botulism, a recently recognized puzzling aspect of the disease, is not classical food poisoning. Therapy of botulism is supportive care following neutralization of the circulating neurotoxin (-NT) with antiserum; the efficacy of antiserum therapy is unclear. There are eight immunologically distinct but pharmacologically similar NT types. The NT specifically blocks quantal release of the neurotransmitter acetylcholine from cholinergic presynapses. The mechanism of this blockade at the molecular level is not known. This proposalis the initial part of a long term project on i) determination of the sequence, study of ii) the structure function relationship and iii) mode of action of botulinum NT. In this three year proposal we plan to work on the following aspects of structure and structure-function relationship: i) Neurotoxin types A, B, E and F (mol. wt. approximately 150,000) will be cleaved enzymatically and/or chemically to generate small numbersof polypeptides of reasonable size. The fragments will be isolated and analyzed to map the homologous and heterologous domains among the neurotoxin types. These fragments will simplify future work on the amino acid sequence. ii) The active center(s), i.e., the minimum structural part of the NT directly involved in toxicity will be delineated in three ways: 1) by selective modification of the cationic (arginine, histidine, lysine, Alpha-NH2), anionic (aspartic, glutamic, C-terminal) and aromatic (tryptophan, tyrosine) groups of the NT and then relating loss of toxicity to the modification reaction; 2) we will treat type E NT with try psin and determine the physiochemical changes of the NT accompanying the resulting greater than 100 fold increase in toxicity; and 3) by identifying amino acid residues involved in binding of NT with ganglioside (GT1b) with concomitant loss of toxicity. The radiolabelled neurotoxin will be essential for future studies (beyond this proposal) on the mode of action of the NT. Therefore, to obtain highly radiolabelled NT with minimum loss in toxicity the amino acid residues not critical for toxicity will be used as targets for radiolabelling through selective modification.