| Project/Area Number |
06506001
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 試験 |
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| Research Field |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | Nagoya University |
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Principal Investigator |
ISOBE Minoru Agricultural Sciences, Nagoya University Professor, 農学部, 教授 (00023466)
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| Co-Investigator(Kenkyū-buntansha) |
HIBI Kiyokatsu Agricultural Sciences, Nagoya Univesity JASCO Co.Ltd., Head of Group, 第2技術部・LC応用技術課, 主任研究員
ICHIBA Ikuko Agricultural Sciences, Nagoya University Associate Professor, 農学部, 助手 (40247680)
ICHIKAWA Yoshiyasu Agricultural Sciences, Nagoya University Associate Professor, 農学部, 助教授 (60193439)
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| Project Period (FY) |
1994 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥39,900,000 (Direct Cost: ¥39,900,000)
Fiscal Year 1996: ¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1995: ¥8,500,000 (Direct Cost: ¥8,500,000)
Fiscal Year 1994: ¥25,000,000 (Direct Cost: ¥25,000,000)
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| Keywords | BIOLUMINESCENCE / DIOXETANONE / protein phosphatase inhibitor / FIREFLY LUCIFERIN / photooxygenation / ultra-sensitive detection / IMMOBILIZED LUCIFERASE / SOUID BIOLUMINESCENCE / ルシフェラーゼの固定化技術 / タンパク脱燐酸酵素阻害剤 / トートマイシン / 発光素子 / 発光タンパク / 蛋白脱燐酸酵素阻害剤 / 発光蛋白 |
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| Research Abstract |
Bioluminescent systems such as firefly luminescenece and squid luminescence were mainly utilized as typical cases requiring luciferase-luciferin (or photoprotein) plus the third factor(s). We have already estalished the technology to use immobilized luciferase of firefly, which has already been produced by E.coli through cloning. The luminescence can be monitored with an equipment similar to HPLC system with modification : thus, pumping a buffer containing ATP and Mg through an autosampler into a column having immobilized luciferase. The light used to be detected with a photomultiplier, and this has been improved to use "single photon counter" with computerized equipment. Thus the data can be transfered to Machintosh and the ordinal detection can be operated at 10^<-14> molar level. The linearity under the condition was max>2x10^<-10> molarlevel.
An application of this system was extended to detect the mode of action of protein phosphatase with its inhibitors such as okadaic acid, tautomycin etc.using both types of phosphatases (type I and II). Okinawan squid produced a specific photoprotein, which was extracted, purified and analized to determine the (partial) amino acid sequence. The lumi-detection system was proved to be useful for these structural studies. Other bioluminescent organisms were also searched in South East Asia with success. On the other hand, great progress was made in the molecular mechanism of luminescence ; thus, we have succeeded in the synthesis (under photooxygenation condition) and spectroscopic detection by ^<13>CNMR and IR of the common intermediate "dioxetanone" at-78ﾟC with ^<13>C enriched luciferin analog. Computer chemistry of the molecular mechanic calculation of the luciferase and luciferin analogs were studied for molecular-molecular interaction.
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