| Project/Area Number |
12045226
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
|
| Allocation Type | Single-year Grants |
|---|
| Review Section |
Science and Engineering
|
|---|
| Research Institution | The University of Electro-communications |
|---|
Principal Investigator |
HARUKI Niwa The University of Electro-communications, Department of Applied Physics and Chemistry, Ptofessor, 電気通信学部, 教授 (20135297)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
MAKI Shojiro The University of Electro-communications, Department of Applied Physics and Chemistry, Asistant Ptofessor, 電気通信学部, 助手 (20266349)
HIRANO Takashi The University of Electro-communications, Department of Applied Physics and Chemistry, Associate Ptofessor, 電気通信学部, 助教授 (20238380)
|
|---|
| Project Period (FY) |
2000 – 2002
|
| Project Status |
Completed (Fiscal Year 2003)
|
| Budget Amount *help |
¥12,600,000 (Direct Cost: ¥12,600,000)
Fiscal Year 2002: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 2000: ¥4,800,000 (Direct Cost: ¥4,800,000)
|
|---|
| Keywords | Latia neritoides / bioluminescence / luciferin analog / structure-activity relationship / luciferase purification / c-DNA / cloning / クローニング / ラチア / ルシフェリン / ルシフェラーゼ / 糖タンパク / アミノ酸配列 / ニュージーランド / エステラーゼ活性 / MALDI-TOF |
|---|
| Research Abstract |
(1) Studies on substrate specificity using a series of analogs of the Latia luciferin suggested that the Latia luciferase recognized strrctly the 2,6,6-trimethylcyclohexene ring moiety in the luciferin structure. The corresponding enol acetate and benzoate analogs also possessed luciferin activities, indicating the formate moiety to be not essential for the Latia bioluminescence reaction. The rate of light production was delayed when the enol acetate analogue and benzoate analogue were used, indicating the hydrolysis step of the enol ester moiety at the active site of the luciferase may be rate determining step of the light production reaction. Interestingly, the corresponding 2,6-dimethylphenyl analogue also have luciferin activity Further studieson the relationship between structure and luciferin activity are currently under investigation.
(2) Purification of Latia luciferase was achieved by a 5-step procedure including affinity chromatography on a Con A-supported column.
(3 ) The purified luciferase exhibited a luciferin/luciferase reaction with the Latia luciferin without any cofactors such as "purple protein," previously reported as the cofactor, and gave the sarne bioluminescence spectra as those obtained with the crude luciferase containing "purple protein," indicating that the light emitter of Latia bioluminescence is a fluorophore present in the luciferase molecule.
(4) The bioluminescent-active luciferase was found to have a molecular mass of 180 kDa and to be present as a hexamer of bioluminescent-inactive monomers having a molecular mass of 31 kDa. The Latia luciferase was found to be a glycoprotein. The glycoside moiety may be important for keeping the oligomeric structure and/or for bioluminescence reaction.
(5) The c-DNA for Latia luciferase was cloned. The primary structure, deduced from the nucleotide sequence, consists of 294 amino acid residue in a single peptide chain with an N-glycosylated NSS sequence.
|
