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Study on Molecular Mechanism of Latia Biohminescnece

拉蒂亚生物化学分子机制研究

基本信息

批准号：
12045226
负责人：
HARUKI Niwa
金额：
$ 8.06万
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 on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

(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.

(1)利用一系列Latia荧光素类似物对底物专一性的研究表明，Latia荧光素酶能准确识别荧光素结构中的2，6，6-三甲基环己烯部分。相应的乙酸烯醇酯和苯甲酸类似物也具有荧光素活性，表明甲酸盐部分对Latia生物发光反应不是必需的。当使用乙酸烯醇酯类似物和苯甲酸酯类似物时，光产生速率被推迟，表明荧光素酶活性部位的烯醇酯部分的水解步骤可能是光产生反应的速率决定步骤。有趣的是，相应的2，6-二甲基苯基类似物也具有荧光素活性。关于结构和荧光素活性之间的关系的进一步研究目前正在进行中。(2)Latia荧光素酶的纯化是通过5步程序实现的，包括在ConA支撑的柱上进行亲和层析。(3)纯化的荧光素酶与Latia荧光素酶发生荧光素/荧光素酶反应，而没有任何辅助因素，如先前报道的辅因子“紫色蛋白”，并给出了与含有“紫色蛋白”的粗品荧光素酶相同的Sarne生物发光光谱，表明Latia生物发光的发光体是荧光素酶分子中存在的一个荧光团。(4)发现具有生物发光活性的荧光素酶的分子质量为180 kDa，并作为分子质量为31 kDa的生物发光不活跃单体的六角体存在。Latia荧光素酶是一种糖蛋白。糖苷部分对于保持低聚结构和/或生物发光反应可能是重要的。(5)克隆了Latia荧光素酶的c-DNA。根据核苷酸序列推导出的一级结构由294个氨基酸残基组成，在一个单肽链上带有N-糖基化的NSS序列。