| Project/Area Number |
61560213
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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 |
General fisheries
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| Research Institution | HIROSHIMA UNIVERSITY |
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Principal Investigator |
NAKAGAWA Heisuke (1987) Faculty of Applied Biological Science, 生物生産学部, 教授 (00034471)
村地 四郎 (1986) 広島大, 生物生産学部, 教授 (40034433)
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| Co-Investigator(Kenkyū-buntansha) |
UEMATSU Kazumasa Faculty of Applied Biological Science, 生物生産学部, 助教授 (00116542)
中川 平介 広島大学, 生物生産学部, 教授 (00034471)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1987: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1986: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | acetylcholine / choline acetyltransferase / fish / electric ray / electric organ / 電気器官 / 抗体 / 免疫組織化学 / コリン作動性神経 / 酵素抗体法 / 魚類神経系 / 酵素精製 / 免疫 |
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| Research Abstract |
In this study, we had tried to purify ChAT of the electric organ of the electric ray Narke japonica to make an antibody to the fish ChAT for staining fish cholinergic nerves immunohistochemically. The ChAT activity was monitored as described by Fonnum (1969) modified by Ryan and McClure (1979) and purification procedure was done essentially according to Ryan and McClure (1979). Two hundred grams of the electric organ of two fish was solubilized and fractionated by precipitation with ammonium sulfate. Acetic acid precipitation in the original method using the mammalian brain was omitted, because the ChAT activity was lost mostly in this step. Further purification was obtained by sequential chromatography on an ion-exchange chromatography with carboxymethyl-Sepharose C-50, on an affinity column of coenzyme A (CoA)-Sepharose and on a hydroxylapatite. In CM-Sepharose column, the emzyme was not absorbed by the column in the buffer of pH 7.2 used in the original method. By a test using buffers of different pH, it became clear that most ChAT was absorbed by gel at pH 6.8. The enzyme was eluted at fractions of about 0.2M NaCl. ChAT was eluted at 0.6-0.8M NaCl in affinity column. In hydroxylapatite column, the enzyme was not only by ammonium sulfate gradient but also by NaCl gradient. Both the fractions contained one main protein of MW 68,000 but not homogeneous. Anti-serum to the fractions was gained in a rabbit and was used for immunohistochemistry of the goldfish spinal cord. However, the anti-serum was not spesific to motoneurones. It was concluded that purification of fish ChAT was as difficult as that of mammals and other methods to stain cholinergic nerves had to be developed. That must be either the production of monoclonal antibodies to ChAT or polyclonal antibodies to ACh itself.
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