High resolution labeling of cholinergic nerve terminals using a specific fully active biotinylated botulinum neurotoxin type A

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Abstract
We report here on the synthesis and characterization of a fully active biotinylated derivative of the botulinum neurotoxin type A. Different ratios of biotin:botulinum toxin were tested to optimize derivatizing conditions and a ratio of 35:1 was selected for further experiments. The average number of biotin groups per toxin molecule was estimated to be 7.8, occurring at both heavy and light chains, and almost all externally located and easily accessible to recognition by streptavidin. The modified toxin retained its toxicity and its ability to interact with biological membranes. Apart from its suitability for detection in Western blots and in microtiter well plates, biotinylated botulinum toxin proved to be adequate for morphological labeling studies at both light and electron microscopy. Peroxidase histochemistry in cryostat sections of intoxicated rat hemidiaphragm muscles showed a distinct labeling of end‐plates. Electron microscopy studies were performed on the electric organ of Torpedo marmorata using colloidal gold‐conjugated streptavidin for detection. After intoxication of electric organ fragments with the modified toxin, gold labels were found associated with the presynaptic plasma membrane of nerve terminals and with the membrane of synaptic vesicles. Moreover, the distribution of biotinylated botulinum toxin binding sites over the membrane of synaptosomes isolated from the electric organ of Torpedo and their relationship with intramembrane particles were analyzed using the replica‐staining label‐fracture technique. It was found that the toxin is never associated with intramembrane particles.