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

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 approximately 26 nM, Bmax approximately 7.5 pmol.mg prot-1; presynaptic plasma membrane: Kd approximately 30 nM, Bmax approximately 52 pmol.mg prot-1). The binding of 125I-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, either 125I-botulinum neurotoxin or botulinum toxin-gold complexes bind to a M(r) approximately 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.

Functional reconstitution of KCl-evoked, Ca2+-dependent acetylcholine release system inXenopus oocytes microinjected with presynaptic plasma membranes and synaptic vesicles

“…This experiment suggests that incorporation of either PSPM or SV into the oocytes provides them with target/s for the action of BoNTiA. A PSPM glycoprotein that binds specifically to BoNT/A has been characterized in our laboratory (Blasi et al, 1992;Canals et al, 1995). Recently, a membrane-associated protein, SNAP-25, has been shown to be cleaved by zinc-protease activity of Botulinum toxin type A (Blasi et al, 1993), as well as, synaptobrevin being cleaved by Tetanus toxin and Botulinum toxin type B (Schiavo et al, 1992).…”

Uptake of Botulinum Neurotoxin into Cultured Neurons

“…. Proteolysis by BoNT-E is blocked much less efficiently such that addition of Con A 20 or 30 min after toxin washout has a weaker effect (9). Both BoNTs show complete insensitivity to Con A 40 min after toxin washout.…”

Botulinum Neurotoxin A Activity Is Dependent upon the Presence of Specific Gangliosides in Neuroblastoma Cells Expressing Synaptotagmin I

“…Also, a 140-kDa protein in cholinergic synaptosomes prepared from Torpedo electric organs was identified as a BoNT/A-binding protein. The interaction of BoNT/A with this 140-kDa protein was both sialidase and trypsin-sensitive (25). These studies among others led to the hypothesis that botulinum neurotoxins require a "double receptor" comprised of protein and gangliosides (26).…”