Binding of botulinum neurotoxin to pure cholinergic nerve terminals isolated from the electric organ of Torpedo

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Abstract
Torpedo electric organ has been used to study the binding of botulinum neurotoxin type A to pure cholinergic synaptosomes and presynaptic plasma membrane.125I-labeled botulinum neurotoxin type A exhibits specific binding to cholinergic fractions. Two binding sites have been determined according to data analysis: a high affinity binding site (synaptosomes: Kd=0.11±0.03 nM, Bmax=50±10 fmol · mg prot−1; presynaptic plasma membrane: Kd=0.2±0.05 nM, Bmax=150±15 fmol · mg prot−1) and a low affinity binding site (synaptosomes: Kd ≈ 26 nM, Bmax ≈ 7.5 pmol · mg prot−1; presynaptic plasma membrane: Kd ≈ 30 nM, Bmax ≈ 52 pmol · mg prot−1). The binding of125I-botulinum neurotoxin type A is decreased by previous treatment of synaptosomes by neuraminidase and trypsin, and by a preincubation with bovine brain gangliosides or antiserum raised against Torpedo presynaptic plasma membrane. When presynaptic plasma membranes are blotted to nitrocellulose sheet, either125I-botulinum neurotoxin or botulinum toxin-gold complexes bind to a Mr ≈ 140,000 protein. Botulinum toxin-gold complexes have also been used to study the toxin internalization process into Torpedo synaptosomes. The images fit the three step sequence model in the pathway of botulinum neurotoxin poisoning.

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