Immunological characterization of papain-induced fragments of Clostridium botulinum type A neurotoxin and interaction of the fragments with brain synaptosomes.
After treatment of Clostridium botulinum type A neurotoxin with papain, three fragments (Mrs, 101,000, 45,000, and 43,000) were purified by hydrophobic and ion-exchange chromatography with a high-performance liquid chromatographic system. Immunoblotting analyses with monoclonal antibodies showed that the 101,000-dalton fragment consisted of the light chain and a part of the heavy chain (H-1 fragment) linked together by a disulfide bond, and the other two fragments were correlated to the remaining portion of the heavy chain (H-2 fragment). The 45,000- and 43,000-dalton fragments effectively competed for binding of the 125I-labeled neurotoxin to synaptosomes, while no inhibition was observed with the 101,000-dalton fragment. The results indicate that the H-2 fragment interacts with the binding site on the neural membrane. The binding of the neurotoxin was impaired by treatment of synaptosomes with neuraminidase. Incorporation of gangliosides into neuraminidase-treated synaptosomes resulted in the restoration of binding. The results suggest that gangliosides are one of the components of the toxin-binding site.
Kozaki
S
S
Department of Veterinary Science, College of Agriculture, University of Osaka Prefecture, Japan.
Miki
A
A
Kamata
Y
Y
Ogasawara
J
J
Sakaguchi
G
G
D000818:Animals; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000276:immunology; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D001905:Botulinum Toxins / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D001921:Brain / Q000378:metabolism*; D003014:Clostridium botulinum / Q000276:immunology*; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D009498:Neurotoxins / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D010206:Papain / Q000494:pharmacology*; D013574:Synaptosomes / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D009498:Neurotoxins; D010206:Papain; D001905:Botulinum Toxins