| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1988: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1987: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Clostridium botulinum neurotoxin acts on nerve endings to inhibit acetylcholine release. The neurotoxin is composed of two fragments, H- and L-chains, held together with at least one disulfide bond. When H-chain was treated with a certain proteolytic enzyme, two smaller fragments were obtained. The neurotoxin molecule, therefore, comprises at least three domains, named L, H-1 and H-2. It has been demonstrated also that the binding of the neurotoxin to the brain synaptosomal membrane is undertaken by H-chain. Monoclonal antibodies (MABS) recognizing H-2 inhibited the binding between the toxin and synaptosomes, while L・H-1 did not. From these results, it can be presumed that H-2 undertakes the binding between the toxin and the neural membrane. After limited digestion of the neurotoxin with papain, H-2 was highly purified by HPLC. Such a highly purified preparation of H-2 was examined for the inhibition of the binding between the neurotoxin and the neural membrane. It inhibited the binding to the same extent as did the intact neurotoxin, proving that it is h-2 that binds the neurotoxin to the neural membrane. Attempts were made to characterize the receptor on the neural membrane that binds the neurotoxin. When treated with neuraminidase, meurotoxin no longer bound to brain synaptosomes. Upon incorporation of gangliosides into the membrane, the binding was partially restored. To the lysylendopeptidase-treated synaptosomes, type A neurotoxin bound, but type B neurotoxin did not. Attempts were made to solubilize the toxin-binding substance under various conditions to isolate the receptor. The solubilized substances were incorporated into liposome and tested for the binding; the same binding pattern as with synaptosomes resulted. From these results, we conclude that gangliosides and proteinous components are associated with the binding between the neurotoxin and the membrane. Purification of the proteinous component is now under way.
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