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Bioluminescence Technology, Development of its Multipul Function

生物发光技术及其多功能性的开发

基本信息

批准号：
06506001
负责人：
ISOBE Minoru
金额：
$ 25.54万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

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.

生物发光系统如萤火虫发光和鱿鱼发光主要用作需要发光酶-荧光素（或发光蛋白）加上第三因子的典型情况。我们已经建立了固定化萤火虫荧光素酶的技术，该荧光素酶已由大肠杆菌通过克隆产生。可以用类似于HPLC系统的设备进行修改来监测发光：因此，将含有ATP和Mg的缓冲液通过自动进样器泵入具有固定化荧光素酶的柱中。光过去是用光电倍增管检测的，现在已经改进为使用带有计算机化设备的“单光子计数器”。因此，数据可以传输到Machinery，并且可以在10 μ mol水平上进行顺序检测<-14>。该系统的线性范围最大> 2 × 10 ~<-10>（-1）mol/L，并应用于蛋白磷酸酶与抑制剂冈田酸、互变霉素等的作用方式的测定。冲绳鱿鱼产生一种特异性发光蛋白，对其进行了提取、纯化和氨基酸序列分析。发光检测系统被证明是有用的，这些结构的研究。在东南亚也成功地搜索了其他生物发光生物。另一方面，发光的分子机理研究也取得了很大进展，我们成功地合成了（在光氧化条件下）<13>-78 ° C的中间体二氧杂环丁烷，并利用~ 13 CNMR和IR进行了光谱检测<13>。本文研究了荧光素酶和荧光素类似物分子间相互作用的分子力学计算的计算机化学。