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Developmental Studies on Ultrasensitive Method for Determination of Polyunsaturated Fatty Acids

多不饱和脂肪酸超灵敏测定方法的进展研究

基本信息

批准号：
04558021
负责人：
KAWAGUCHI Akihiko
金额：
$ 4.67万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04558021/
关键词：
Polyunsaturated fatty acids Acyl-CoA synthetase Acyl-CoA oxidase Multienzyme complex involved in fatty acid oxidation cDNA 高度不飽和脂胞酸

项目摘要

The object of this research is to develop a new method to analyze polyunsaturated fatty acids quantitatively using acyl-CoA synthetase (ACS), acyl-CoA oxidase (ACO) and multienzyme complex involved fatty acid beta-oxidation (HDT). We established over-expression systems of ACO and HDT and purified these enzymes. The major problem of this project is to isolate ACS which is specifically active to polyunsaturated fatty acids. We isolated five different species of ACS (ACS-I,II,III,BACS-I,II) cDNAs from rat and investigated their structures and properties. ACS-I and ACS-II were mainly expressed in liver and ACS-III was in intestine. On the other hand, both BACS-I and BACS-II were predominantly expressed in brain. ACS-I,ACS-III and BACS-I showed similar specificity to fatty acids. On the other hand, ACS-II and BACS-II showed different fatty acid specificity from ACS-I,ACS-III and BACS-I.BACS-II,which was expressed in COS cells, efficiently utilized eicosapentaenoic acid, arachidonic acid, myristic acid and lauric acid. ACS-II,which was expressed in E.coli, exhibited the highest activity to eicosapentaenoic acid, arachidonic acid and docosahexenoic acid.From these results, ACS-II is the most suitable ACS to determine polyunsaturated fatty acids. The bacterial ACS,which we have previously obtained, is reactive to saturated fatty acids and unsaturated fatty acids with one or two double bonds but not to eicosapentaenoic acid, arachidonic acid and docosahexenoic acid. It is possible to determine eicosapentaenoic acid, arachidonic acid and docosahexenoic acid with the combination of ACS-II and bacterial ACS since ACS-II can slightly activate saturated fatty acids.

本研究的目的是建立一种利用酰基辅酶A合成酶(ACS)、酰基辅酶A氧化酶(ACO)和涉及脂肪酸β-氧化的多酶复合体(HDT)来定量分析多不饱和脂肪酸的新方法。我们建立了ACO和HDT的高效表达系统，并对这些酶进行了纯化。本项目的主要问题是分离出对多不饱和脂肪酸具有特异性活性的ACS。我们从大鼠中分离了五种不同种类的ACS(ACS-I，II，III，BACS-I，II)的cDNA，并对其结构和性质进行了研究。ACS-I和ACS-II主要在肝脏表达，而ACS-III主要在肠道表达。另一方面，BACS-I和BACS-II主要在脑内表达。ACS-I、ACS-III和BACS-I表现出与脂肪酸类似的特异性。在COS细胞中表达的BACS-II能有效利用二十碳五烯酸、花生四烯酸、肉豆蔻酸和月桂酸。在大肠杆菌中表达的ACS-II对二十碳五烯酸、花生四烯酸和二十二碳六烯酸的活性最高，是最适合测定多不饱和脂肪酸的ACS。我们以前获得的细菌ACS能与饱和脂肪酸和带有一个或两个双键的不饱和脂肪酸反应，但不能与二十碳五烯酸、花生四烯酸和二十二碳六烯酸反应。由于ACS-II对饱和脂肪酸有轻微的激活作用，因此可以用ACS-II和细菌ACS相结合的方法测定二十碳五烯酸、花生四烯酸和二十二碳六烯酸。