Botulinum neurotoxin (MT), produced by the bacteria Clostridium Botulinum, is the sole cause of the dreaded neuroparalytic disease botulism. The NT blocks release of the neurotransmitter acetylcholine (ACh) at peripheral cholinergic synapses. Consequently flaccid muscle paralysis occurs. This property of the NT is being exploited as an experimental drug for certain spastic muscle conditions, e.g. blepharospasm, hemifacial spasm, and strabismus in human patients. The NT is thought to act in three steps; it binds to nerve membrane, then moves inside (translocation) and finally blocks ACh release; mode of action at the molecular level is unknown. This proposal is part of a long- term study of the mechanism of action of the NT protein (Mr 150,000) which is composed of two subunit chains, H and L (Mr 100,000 and 50,000, respectively). We plan to locate and partially characterize the segments of this large protein involved in these three steps in the following ways: 1) Experiments with isolated H chain and neuromuscular junction (NMJ, from mouse phrenic hemidiaphragm) will confirm that the binding site of NT is on the H chain. 2) The H chain will be enzymatically cut into two halves (each of Mr ~50,000) to determine if the binding site is located on the N-terminal or C-terminal half. 3) Narrower segment of the H chain that encloses the binding site will be characterized biochemically to examine if the binding sites of antigenically distinct NT types A, B and E are identical/dissimilar. 4) Receptors on NMJ for NT types A, B, E will be examined, are they similar? 5) The isolated L chain causes paralysis only when H chain is bound to NMJ. Therefore exclusively radiolabeled L or H chain will be used to determine if one or both chains cross the presynaptic membrane (translocation). 6) Since isolated H chain bound to NMJ does not cause paralysis, which segment of H chain mediates the paralytic activity of the L chain and its presumed entry into the membrane? For this question the two enzymatic fragments of H chain will be examined. 7) Attempts will be made to insert the isolated L chain into the presynaptic membrane via liposome vesicles circumventing the binding step (mediated by the H chain). Success in inducing paralysis will argue that the H chain of dichain NT presents the L chain to the target site and paralysis results only after L chain is inserted into the membrane. 8) The segment of L chain responsible for neurotoxicity will be located, isolated and characterized by enzymatic fragmentation and selective modification of amino acid residues.
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