| Co-Investigator(Kenkyū-buntansha) |
NISHIMURA Masakazu University of Osaka Prefecture, College of Agriculture, 農学部, 助教授 (50011995)
KAMATA Yoichi University of Osaka Prefecture, College of Agriculture, 農学部, 助手 (20152837)
KOZAKI Shunji University of Osaka Prefecture, College of Agriculture, 農学部, 助手 (10109895)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1986: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1985: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Clostridium botulinum neurotoxin types A through G acts presynaptically on nerve endings to inhibit acetylcholine release. The neurotoxin is composed of two fragments, the heavy chain(H) of 100K and the light chain(L) of 50K. For the toxic action, these two fragments function complementarily. We analyzed the fine antigenic structure and the function of the neurotoxin with monoclonal antibodies and demonstrated that the neurotoxin molecule comprises at least three fragments named L, H-1 and H-2. The site of binding to the brain synaptosomal membrane was located on H-2, which is located at the C-terminus of the neurotoxin molecule. The structure of the ganglioside the toxin binds, which is regarded as a receptor candidate, was located on domains differed from one type to another. Type E neurotoxin bound to ganglioside <G_(Q1b)> , whereas type B hardly did so. L・H-1 fragment lacking H-2 did not bind to any ganglioside, but to cerebroside and free fatty acids composing gangliosides. These
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results suggest that H-2 binds to the sugar chain, forming the hydrophilic portion of ganglioside, and that L・H-1 fragment, particularly H-1 binds to the hydrophobic portion. H-2 binds to the receptor on the surface of the neural membrane and H-1 plays an important role in the toxin internalization. By use of mouse antineurotoxin antibody and gold colloid-labelled anti-mouse IgG, toxin molecules were located under an electron microscope at the mouse myoneural junction of the mice exposed to the toxin in vivo and in vitro. Gold colloid was detected on the external membrane of the myoneural junction, invaginated parts of the membrane, in the synaptic vesicles, and the cytoplasm of the ending. The toxin translocates into the nerve endings in association with the formation of synaptic vesicles, followed by internalization into the cytoplasm. Further studies are in progress to correlate the function of each fragment of the toxin found by analysis with monoclonal antibodies and the localization of the toxin at the nerve endings. Less
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