| Project/Area Number |
60440025
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
General physiology
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| Research Institution | Kumamoto University |
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Principal Investigator |
SHIGA Kiyoshi Kumamoto University Medical School (Professor), 医学部, 教授 (40028527)
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| Co-Investigator(Kenkyū-buntansha) |
TOUZYOU Hiromasa Osaka University Medical School (Assistant Professor), 医学部, 助手 (90135707)
WAKITA Yoshiakira Kumamoto University Medical School (Associate Professor), 医学部, 助手 (80040179)
NISHINA Yasuzo Kumamoto University Medical School (Associate Professor), 医学部, 助教授 (50112553)
佐藤 恭介 熊本大学, 医学部, 助手 (80205905)
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| Project Period (FY) |
1985 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 1988: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1987: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1986: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1985: ¥6,000,000 (Direct Cost: ¥6,000,000)
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| Keywords | flavin / amino acid oxidase / 分子間相互作用 / フラビン酵素 / セミキノン / 共鳴ラマン分光 / フラビン蛋白 / ビタミン【B_2】 / アミン酸酸化酵素 / 分子集合形成機構 / 酸化還元酵素 / アミノ酸 / ラマンスペクトル |
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| Research Abstract |
Picolinate binds to the anionic semiquinoid form of D-amino acid oxidase (DAO), and the complex formed has a broad absorption band in the long-wavelength region extending beyond 800 nm, which is reminiscent of a charge transfer interaction. The binding has a stoichiometry of 1:1 with respect to the enzyme. The dissociation constant at 25゜C was 30 M at pH7.0 .The ph dependence (pH7.0-8.3) of the dissociation constant indicates that one proton is associated with the complex formation, and suggessts that picolinate able to bind to the anionic semiquinoid enzyme is in the cationic form protonated at the nitrogen atom. By adding dithionite to the oxidized DAO solution containing pyruvate and various amines, a similar anionic semiquinoid DAO complex having a broad long-wavelength absorption band, appeared. Resonance Raman spectra with excitation at 623.8nm of the anionic semiquinoid DAO complex formed in the presence of pyruvate and methy lamine indicate that the complex consists of the anionic semiquinoid DAO and N-methyl- -iminopropionate produced from pyruvate and methylamine, and that the imino group must be protonated. This supports the proposal that the presence of a positively charged group in the vicinity of flavin is required for the stabilization of the anionic semiquinoid flavin.The results also suggest that the broad absorption band is derived from the charge transfer interaction between the anionic semiquinoid flavin and the imino acid, in which the flavin
C(4a)-N(5) locus and the locus containing H-N^^+__=C-COO^- of the amino acid are important for
the interaction. The charge-transfer complexes of anionic semiquinoid DAO hardly react with a reducing agent (dithionite or photoreduction) or an oxidizing agent (oxygen), different from free anionic semiquinoid DAO. These phenomena can be explained by the complexation (charge-transfer complex) with ligands over the flavin C(4a)-N(5)locus.
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