Summary of Research Project Results Under the JSPS FY2000
"Research for the future Program"



1.Research Institution Nagoya University
 
2.Research Area Life Sciences
 
3.Research Field Structure and Functional Control Mechanism of Biomolecules
 
4.Term of Project FY1996〜FY2000
 
5.Project Number 96L00504
 
6.Title of Project Structure Recognition and Molecular Mechanism in Bioluminescence

7.Projetct Leader
Name Institution,Department Title of Position
Minoru Isobe Nagoya University, Graduate School of Bioagricultural Sciences Professor.

8.Core Members

Names Institution,Department Title of Position
Tsukasa Matsuda Nagoya University, Graduate School of Bioagricultural Sciences Professor
Akira Takai Nagoya University, Graduate School of Medicine Assistant Professor

9.Cooperating Researchers

Names Institution,Department Title of Position
Hidenori Kai Tottori University, Faculty of Agriculture Professor

10.Summary of Research Results

The photoprotein, symplectin, was obtained from Okinawan squid responsible for bioluminescence, and its 501 amino acids were sequenced through Q-TOF-MS analysis as well as cDNA method.
Light production was confirmed by overexpression of this protein through cloning to E. coli. The chromophore that include 100% 13C labeling and several analogs were synthesized and it was recombinated with aposymplectin to provide luminescence, and the molecular mechanism of the luminescent process has been elucidated. Thus, the chromophore binds to protein via covalent bond through a sulfur atom of cystein residue. The recombinant symplectin with a fluorine-containing chromophore afforded evidence of a fluorescent peptide through nano-LC-Q-TOF-MS/MS method to elucidate that the 340 cystein might bind the chromophore. Site specific modification of porteins can be readily monitored by this new methodology, which made protein analysis and modified protein analysis very easy. On the other hand, the analytical method established during this project was applied to other proteinaous system; thus, time interval measurement enzyme is a glycoprotein comprising from 156 amino acid residue with 4 sugar chains through aspargine-22 through N-glycoside linkage.

11.Key Words

(1)luminous squid、(2)symplectin、(3)cDNA cloning
(4)13C-NMR、(5)chromophore、(6)nano-LC-Q-TOF-MS
(7)EA4、(8)tryptic digestion、(9)glycoprotein

12.References

[Reference Articles]
Author Title of Article
Kuse, M.; Isobe, M. Synthesis of 13C-Dehydrocoelenterazxine and NMR Studies on Bioluminescence of Symplectoteuthis model
Journal Volume Year Pages Concerned
Tetrahedron 56 2000 2629-2639

Author Title of Article
Takai, A.; Tsuboi, K.; Koyasu, M.; Isobe, M Effects of modification of the hydrophobic C1-C16 segment of tautomycin on its affinity to type 1 and type 2A protein phosphatases
Journal Volume Year Pages Concerned
Biochemical Journal 350 2000 81-88

Author Title of Article
Liu, Tong-Zhu; Isobe, M. Synthetic Studies on the HIJK-Ring Fragment of Ciguatoxin
Journal Volume Year Pages Concerned
Tetrahedron 56 2000 5391-5404

Author Title of Article
Suwan, S.; Isobe, M.; Kanokmedhakul, S.; Lourit, N.; Kanokmedhakul, K.; Soytong, K.; Koga, K. Elucidation of Great Micro-heterogeneity of an Acidic-Neutral Trichotoxin Mixture from Trichoderma harzianum by ESI-QTOF Mass Spectrometry
Journal Volume Year Pages Concerned
J. Mass Spectrometry 35 2000 1438-1451

Author Title of Article
Kuse, M.; Suwan, S.; Koga, K.; Franz, T.; Kanakubo, A.; Isobe, M.; Shimomura, O. 7, 8-Dihydropterin-6-carboxylic Acid as Light Emitter of Lumonous Millipede, Luminodesmus sequoiae
Journal Volume Year Pages Concerned
J. Bioorganic Chem. Lett.   2001  

Author Title of Article
Tani, N.; Kamada, G.; Ochiai, K.; Isobe, M.; Suwan, S.; Kai. H. Carbohydrate Moiety of Time-Interval Measuring Enzyme Regulates Time Measurement through Its Interaction with Time-Holding Peptide PIN
Journal Volume Year Pages Concerned
J. Biochem. 129 2001 221-227

[Reference Books]
Author Title of Book
Isobe, M. Bioscience, Biotechnology, Biochemistry
Publisher Year Pages
the Japan Society for Bioscience, Biotechnology, and Agrochemistry 2000 12


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