Binding of botulinum neurotoxin to pure cholinergic nerve terminals isolated from the electric organ of Torpedo.
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.
Blasi
J
J
Laboratori de Neurobiologia Cellular i Molecular, Hospital de Bellvitge, Spain.
Egea
G
G
Castiella
M J
MJ
Arribas
M
M
Solsona
C
C
Richardson
P J
PJ
Marsal
J
J
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001905:Botulinum Toxins / Q000378:metabolism*; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003102:Colloids; D004557:Electric Organ / Q000294:innervation*; D006046:Gold; D007106:Immune Sera / Q000276:immunology; D008854:Microscopy, Electron; D009411:Nerve Endings / Q000378:metabolism*; D010275:Parasympathetic Nervous System / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D013569:Synapses / Q000276:immunology / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism; D014101:Torpedo
D003102:Colloids; D007106:Immune Sera; D011956:Receptors, Cell Surface; D006046:Gold; D001905:Botulinum Toxins; D000109:Acetylcholine