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

Summary

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: K_d=0.11±0.03 nM, B_max=50±10 fmol · mg prot⁻¹; presynaptic plasma membrane: K_d=0.2±0.05 nM, B_max=150±15 fmol · mg prot⁻¹) and a low affinity binding site (synaptosomes: K_d ≈ 26 nM, B_max ≈ 7.5 pmol · mg prot⁻¹; presynaptic plasma membrane: K_d ≈ 30 nM, B_max ≈ 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 M_r ≈ 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.