1988 Fiscal Year Final Research Report Summary
Analysis of the molecular mechanisms of neurotransmission by botulinum neurotoxin
| Project/Area Number |
62560286
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
基礎獣医学
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| Research Institution | Hokkaido University |
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Principal Investigator |
SYUTO Bunei Fact. of Vet. Med., Hokkaido Univ., 獣医学部, 助手 (60001533)
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| Co-Investigator(Kenkyū-buntansha) |
KUBO Shuichiro Fact. of Vet. Med., Hokkaido Univ., 獣医学部, 教授 (40001515)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Botulinum toxin / ADP-ribosylation / 神経伝達 / 毒素結合物資 |
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| Research Abstract |
The following informations were obtained in the studies on the botulinum toxins and their functions.1) On the structure and biological activities of the toxin molecules. Botulinum C1 and D toxins were roughly classified into 4 subtypes by the antigenic characteristics. However, the primary structures of the toxins were different within the same type. Moreover, the biologiacl activities (lethality, inhibition of transmitter release, neurocytotoxicity and ADP-ribosylation of GTP binding proteins) of botulinum toxin also differed from each other. Therfore, C1 and D botulinum toxins have strain specificities in the structure and activities. The relationships between these activities are still obscure. 2) In the limited digestion with papain the toxin molecule was cleaved into two fragments with a molecular weight of 100,000 and with a 50,000. The former enhanced the lethality of the mother toxin and the latter inhibited. The binding of toxin to synaptozomes was inhibited with 50,000 fragme
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nt. The analysis of the functional structure by using monoclonal antibodies against the toxin molecule revealed that botulinum toxin molecule consists of three domains for the binding, the translocating and for the acting. 3) On the toxin binding substances and toxin function. Toxin-bound synaptosomes were solubilized with Triton X-100. The solubilized fraction was applied on an affinity column conjugated with anti-toxin monoclonal antibody and the toxin-toxin binding substance complexes were separated. Three kinds of protein molecules with molecular weights of 80,000, 50,000 and 25,000 were detacted by SDS-polyacrylamide gel electrophoresis. One of them (25,000 m.w) may correspond to a substrate of ADP-ribosylation since two kinds of proteins with molecular weights of 24,000 and 26,000 were ADP-ribosylated in the primary cultured cells from rat brain. Toxin inhibited the release of transmitters from the primary cultured cells, but the inhibition was not cancelled by digitonin treatment. Toxin action may be independent on Ca influx. Less
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