Cleavage of intracellular substrates of botulinum toxins A, C, and D in a mammalian target tissue

The objective of the current study was to determine whether the intracellular targets that mediate the mechanism of action of botulinum toxin at the mammalian neuromuscular junction are the same as those identified in nontarget tissues. Previous studies of this nature have been limited to nontarget tissues because of the perceived low abundance of neural proteins in a neuromuscular preparation. In the current study we have used differential centrifugation to concentrate neural proteins in a synaptosomal-enriched fraction from the mouse phrenic nerve-hemidiaphragm preparation. Immunoblot detection re-vealed the presence of discrete immunoreactive bands corre-sponding to SNAP-25, synaptobrevin II, and syntaxin I in the innervated region of the neuromuscular preparation. The ability of these proteins to serve as botulinum toxin substrates i