Clostridial neurotoxins proteolytic activity

Fig. 2. Schematic drawing that summarizes the procedure for monitoring the blockade of neurotransmitter release from brain synaptosomes, followed by immu-noblot analysis to detect specific proteolytic activity of clostridial neurotoxins on poisoned synaptosomes...

In vitro Assay of L Chain Activity. Since the elucidation of the molecular mechanisms of all clostridial neurotoxins, it has become convenient to test the proteolytic activity of the L chain in vitro. For this purpose, L chain or reduced di-chain toxin is incubated with substrate protein. The cleavage products are then separated by SDS-PAGE and analyzed by immunoblotting or autoradiography. 

If there is any problem with protein detection, enriched synaptic protein preparations (synaptosomes, presynaptic plasma membrane of synaptic vesicle preparations, depending of the toxin and the substrate) can be better starting material than brain homogenate to check the proteolytic activity of clostridial neurotoxins L chains. 

Demonstration of this activity was initially difficult because the toxins are very specific for their substrates. However, it is now clear that (a) the clostridial neurotoxins express proteolytic activity and (b) this activity is absolutely required for toxicity.30 The substrate proteins for this action appear to be part of a hetero-oligomeric assembly associated with the synaptic vesicles. Interestingly, the specific target site for cleavage seems to be different for each serotype of the botulinum toxins in some cases, different locations on the same protein in others, different proteins of the assembly.30 The basis of this marked specificity is not yet clear and remains fascinating to scientists interested in neurosecretion. 

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