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Analysis of structure-function correlation of oryzacystatin and development of new functional foods

谷胱抑素结构功能相关性分析及新型功能食品开发

基本信息

批准号：
13460054
负责人：
TANOKURA Masaru
金额：
$ 3.78万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Oryzacystatin (OC) contained within rice (Oryza sativa) is the protein, which inhibits cysteine protease (CP) specifically. It inhibits papain stoichiometrically and has a great thermostability, its activity still remain even if under the conditions of cooking rice, and also is stable with acid and alkali. It regulates the metabolism of seed by controlling the activity of CP, which is concerned with protein degradation in seed. OC also recognizes the CP that exists in foreigners or sources of infection like insect, bacteria and virus as an extrinsic target enzyme and inhibits their activity, so it is said to play an important role in biological defense.This study is focused on function-structure correlation analysis of OC using biochemical method and three-dimensional structural analysis. We succeeded in conversion between dimer and monomer of OC freely by controlling the temperature and pH of solution or concentration of protein or by addition of denaturing agent. We revealed that dimer has less inhibitory activity than monomer. This result suggests that OC acts as a regulator, which regulates protease activity by its conformational change depending on various environmental changes in the stages of development of rice. Solution NMR structure of OC revealed the conformational change occurred with the regions of the first loop and the second loop, which are important for inhibition activity, when it is dimerized. This change would be the cause of the decrease of inhibition activity. In addition, we succeeded in crystallization with homodimer of OC and diffraction data was collected to 2.9 A.OC is applicable to drug medicine and functional food. It became clear that the activity of OC could be regulated by change the solution condition in this study, so we can use this mechanism for construction of nanomachine controlled by pH, and have resistance to acid and heat.

水稻半胱氨酸蛋白酶抑制剂（Oryzacystatin，OC）是水稻中特异性抑制半胱氨酸蛋白酶（cysteine protease，CP）的蛋白质。它对木瓜蛋白酶有化学计量的抑制作用，并具有很好的热稳定性，即使在煮米饭的条件下，其活性仍然存在，对酸和碱也很稳定。它通过控制CP的活性来调节种子的代谢，而CP的活性与种子中蛋白质的降解有关。OC还将存在于外来物或传染源（如昆虫、细菌和病毒）中的CP识别为外源靶酶，并抑制其活性，因此被认为在生物防御中发挥重要作用。通过控制溶液的温度、pH值、蛋白质浓度或加入变性剂，成功地实现了OC二聚体与单体的自由转化。我们发现，二聚体的抑制活性低于单体。这一结果表明，OC作为一种调节剂，它调节蛋白酶活性的构象变化，这取决于不同的环境变化，在水稻的发展阶段。溶液NMR结构分析表明，OC二聚化后，对抑制活性起重要作用的第一环和第二环区域发生了构象变化。这种变化可能是抑制活性下降的原因。此外，我们还成功地用OC的同二聚体进行了结晶，衍射数据收集到2.9埃。OC适用于药物、医药和功能食品。本研究表明，OC的活性可以通过改变溶液条件来调节，因此可以利用这一机制来构建pH可控的纳米机器，并具有耐酸性和耐热性。