Binding of Clostridium botulinum Type C Neurotoxin to Rat Brain Synaptosomes1
Journal of Biochemistry (Tokyo)
The binding of Clostridium botulinum type C neurotoxin to the synaptosomes was examined by two methods, by determination of the toxicity remaining in the supernate after incubation of the reaction mixture, and by enzyme-linked immunosorbent assay (ELISA) with the bound toxin in the precipitate. 1) By ELISA in the microtiter plate system, 10-1000 ng of toxin could be determined by the use of rabbit anti-(botulinum toxin type C) IgG and goat anti-(rabbit IgG) IgG-fi-galactosidase complex. 2) The
... at brain synaptosomes were stored at -80°C in 10 % dimethylsulfoxide for three months. The synaptosomes retained 70% of the activity of He-choline uptake in the fresh preparation and maintained the K+ -stimulated release of HC-acetylcholine and the binding capacity to the toxin. However, the synaptosomes stored at O°C resulted in an impairment of choline uptake for a period of one week. 3) More than 94 % of the toxin (5.3 x 10 6 MLD/ml, 220 pg/ml) was bound when incubated at O°C with the synaptosomes (2 mg protein/ml) and then measured by the amount of toxicity remaining in the supernate of the reaction mixture. Although the binding was equilibrated within 5 min, the binding amount of toxin to the synaptosomes determined by ELISA was less than 1 % of that obtained by the former method. 4) The dose dependence of the toxin to the synaptosomes showed the typical curvilinear curve proposed by STECK & WALLACH (1965). This finding suggests that there are two kinds of toxic receptors on the synaptic membrane which show a difference of association constant and that the number of receptors per 1 mg of synaptosomal protein are 4.7 x 1011 M-l and 3.38 x 10 15 , and 5.0 x 10 8 M-I and 4.63 x 10 16 , respectively. 5) The binding amount of heavy and light chains of the toxin to the synaptosomes was less than that of the parental toxin. However, the bound heavy chain was a thousand times higher than that of the light chain, suggesting that the binding site of the toxin may be located in the heavy chain.