1987 Fiscal Year Final Research Report Summary
ACTION OF BOTULINUM NEUROTOXIN ON ACETYLCHOLINE RELEASE FROM SYNAPTOSOMES
| Project/Area Number |
61480080
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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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 |
KUBO SHUICHIRO HOKKAIDO UNIVERSITY, FACULTY OF VETERINARY NEDICINE.PROFESSOR, 獣医学部, 教授 (40001515)
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| Co-Investigator(Kenkyū-buntansha) |
SYUTO BUNEI HOKKAIDO UNIVERSITY, FACULTY OF VETERINARY MEDICINE. ASSISTANT PROFESSOR, 獣医学部, 助手 (60001533)
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| Project Period (FY) |
1986 – 1988
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| Keywords | Boutulinum nerotoxin / Neurotransmitter system / Inhibition of acetylcholine release / Synaptosomes / Development mechanism of toxicity / 細胞内侵入 / 細胞内代謝応答 / ADP-リボシル化反応 |
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| Research Abstract |
THE RELATIONSHIP BETWEEN THE INHIBITION OF ACETYLCHOLINE RELEASE AND THE BINDING AND INTERNALIZATION OF THE TOXIN WAS STUDIED USING RAT BRAIN SYNAPTOSOMES.
THE TOXIN BOUND TO SYNAPTOSOMES REVERSIBLY THROUGH THE HEAVY CHAIN IN THE INCUBATION AT 0゜C. HOWEVER, THIS REVERSIBILITY DECREASED WITH INCREASING THE INCUBATION TEMPERATURE AND TIME AND COMPLETLY LOST IN THE INCUBATION AT 37゜C, SUGGESTING THE FORMATION OF THE CLUSTERING OF TOXIN-SYNAPTOSOMES-COMPLEX. IN THIS BINDING, GANGLIOSIDES IN THE SYNAPTOSOMAL MEMBRANE ACT CO-OPERATELY TO THE TOXIN WITH OTHER POTENTIAL RECEPTOR(S) SUCH AS THE BLYCOPROTEIN. IF THE TOXIN WAS REACTED WITH N-OXYL-IMIDAZOLE, 3 MOLES OF TYROSINE RESIDUES WAS MODIFIED FAST WITH THE REAGENT AND THE TOXIN DECREASED MARKDLY ITS BINDING ABILITY TO SYNAPTOSOMES, SUGGESTING THESE TYROSINE RESIDUES INVOLVE IN THE BINDING OF THE TOXIN AND SYNAPTOSMES.
THE MAXIMUM INHIBITION OF [^<14>C]ACETYLCHOLINE RELEASE FROM THE SYNAPTOSOMES WAS OBSERVED AFTER 15 MIN (LAG TIME) PRE-INCUBAT
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ION AT 37゜C. THE TOXIN-BINDING-SYNAPTOSOMES WERE REACTED WITH ANTI-TOXIN SUBUNITS Fabs AT 0゜C FOR 1.5 MIT BEFORE PRE-INCUBATION AT 37゜C. BOTH Fabs REVERSED THE ACETYLCHOLINE RELEASE INHIBITION BY THE TOXIN. HOWEVER, WHEN THE Fabs WERE ADDED DURING THE PRE-INCUBATION TIME AT 37^C THEY' SHOWED LESS RESTORATION WITH INCREASING PRE-INCUBATION TIME. THE RESTORATION WAS COMPLETELY ABOLISHED IF THE Fabs WERE ADDED TO THE SYNAPTOSOMES AFTER THE FIRST HALF OF THE "LAG TIME". ON THE OTHER HAND, WHEN ^<125>I-TOXIN-BINDING-SYNAPTOSOMES WERE REACTED WITH Fabs BEFORE PRE-INCUBATION OF THE SYNAPTOSOMES, ANTI- HEAVY CHAIN Fab REMOVED ^<125>I-TOXIN FROM THE SYNAPTOSOMES, BUT ANTI-LIGHT CHAIN Fab DID NOT. IF THE Fabs WERE ADDED TO TOXIN-BINDING-SYNAPTOSOMES DURING THE PRE-INCUBATION TIME AT 37゜C, THE Fabs COULD NOT REMOVED ^<125>I-TOXIN FORM THE SYNAPOTOSMES, AND THE SYNAPTOSOMES RETAINED MORE LABELED TOXIN WITH INCREASING PRE-INCUBATION TIME.
THESE RESULTS SUGGEST THAT THERE ARE THREE STEPS (BINDING, INTERNALIZATION, LYTIC) IN THE INHIBITION OF ACETYLCHOLINE RELEASE FROM SYNAPTOSOMES BY THE TOXIN. Less
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