Study on Molecular Mechanism of Sea Firefly Bioluminescence : Elucidation of Acitive Site in the Luciferase

海萤生物发光的分子机制研究：荧光素酶活性位点的阐明

• 批准号: 10680562
• 负责人: NIWA Haruki
• 金额: $ 2.11万
• 依托单位: The University of Electro-communications
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680562/
• 关键词: New Zealand; luminous organism; Latia neritoides; luciferin; luciferase; m-RNA; c-DNA; cloning; ウミホタル; MALDI-MS

1) In connection with the structure characterization of post-transrationaly modified proteins by means of mass spectrometry, matrix asisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry was applied to differentiation between a pair of glycosidation site isomeric oligosaccharides, and to determine the phosphorylation site of phosphpeptides. The glycosidation site isomers were found to be distinguished from each other on the basis of post source decay (PSD) spectra from spesific precoursor ions. We found also MALDI-TOF/in source decay (ISD) mass spectrometry to be powerful method for determination of the phosphorylation site of phosphopeptides.2) We developed a MALDI/TOF mass method for small organic molecules using an ioorganic particle matrix suspended in a viscous dispersant such as liquid paraffin.3) A photoreactive coelenterazine analogue having an azide group was synthesized, and was found to show similar chemi- and bioluminescence properties to those of natural coelenterazine. Photolysis of the synthesized coelenterazine analogue in the presence of diethylamine gave a azepine derivative derived from the generated nitrene species, as the major product. photoreactive coelenterazine analophotoreactive coelenterazine analo.4) The small marine ostracod crustacean Vargula hilgendorfi luciferase was purified from the gland-ejected luciferin and luciferase solution in seawater by using size-exclusion chromatography. The luferase was found to be a glycoprotein, which has a molecular mass of 61.7 kDa and pI values of 3.9-5.0, and is a sort of heat stable protein, which maintained 80% activity when incubated for 10 hr at 37 ℃. The activities of the enzyme were maintained in the presence of 0.1 M NaCl, KCl, and MgCl_2.

1）结合质谱法对转导后修饰蛋白的结构表征，采用基质辅助激光解吸电离（MALDI）-飞行时间（TOF）质谱法区分一对糖苷化位点异构寡糖，并确定磷酸化肽的磷酸化位点。根据特定前体离子的源后衰变 (PSD) 光谱，发现糖苷化位点异构体彼此不同。我们还发现 MALDI-TOF/源衰变 (ISD) 质谱法是测定磷酸肽磷酸化位点的有力方法。2) 我们开发了一种用于小有机分子的 MALDI/TOF 质量方法，使用悬浮在粘性分散剂（如液体石蜡）中的有机颗粒基质。3) 具有叠氮基团的光反应性腔肠素类似物是 合成的，并被发现表现出与天然腔肠素相似的化学和生物发光特性。合成的腔肠素类似物在二乙胺存在下光解，得到源自生成的氮宾物种的吖庚因衍生物，作为主要产物。光反应性腔肠素analophotoreactive腔肠素analo。4)通过尺寸排阻色谱从腺体排出的荧光素和海水中的荧光素酶溶液中纯化小型海洋介形类甲壳动物Vargula hilgendorfi荧光素酶。荧光素酶是一种糖蛋白，分子量为61.7 kDa，等电点为3.9-5.0，是一种热稳定性蛋白，37℃孵育10小时仍能保持80%的活性。在 0.1 M NaCl、KCl 和 MgCl_2 存在下，该酶的活性得以维持。

项目成果

Jing Ling Zheng ら: "Synthesis and Chemi- and Bio-luminescence Properties of a Photolabile Coelenterazine Analogue Bearing an Azido Group."Bull.Chem.Soc.Jpn.. 73. 465-469 (2000)

Jing Ling Cheng 等人：“带有叠氮基的光不稳定腔肠素类似物的合成及其化学和生物发光特性。Bull.Chem.Soc.Jpn.. 73. 465-469 (2000)

Jing Ling Zheng, Feng Qi Chen, Takashi Hirano, Yoshihiro Ohmiya, Shojiro Maki, Haruki Niwa, and Mamoru Ohashi: "Synthesis and Chemi- and Bio-luminescence Properties of a Photolabile Coelenterazine Analogue Bearing an Azido Group."Bull.Chem.Soc.Jpn.. 73, N

Jing Ling Cheng、Feng Qi Chen、Takashi Hirano、Yoshihiro Ohmiya、Shojiro Maki、Haruki Niwa 和​​ Mamoru Ohashi：“带有叠氮基团的光不稳定腔肠素类似物的合成及其化学和生物发光特性。”Bull.Chem.Soc