Developmental Studies on Ultrasensitive Method for Determination of Polyunsaturated Fatty Acids

本研究旨在建立一种利用酰基辅酶a合成酶（ACS）、酰基辅酶a氧化酶（ACO）和脂肪酸β -氧化多酶复合物（HDT）定量分析多不饱和脂肪酸的新方法。我们建立了ACO和HDT过表达系统，并纯化了这两种酶。本课题的主要问题是分离对多不饱和脂肪酸具有特异性活性的ACS。我们从大鼠体内分离出5种不同的ACS (ACS- i，II，III,BACS-I，II) cdna，并研究了它们的结构和性质。ACS-I和ACS-II主要表达于肝脏，ACS-III主要表达于肠道。另一方面，BACS-I和BACS-II在大脑中主要表达。ACS-I、ACS-III和BACS-I对脂肪酸具有相似的特异性。另一方面，ACS-II和BACS-II表现出与ACS-I、ACS-III和BACS-I不同的脂肪酸特异性。BACS-II在COS细胞中表达，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。综上所述，ACS- ii是测定多不饱和脂肪酸最合适的ACS。我们之前获得的细菌ACS对饱和脂肪酸和带有一个或两个双键的不饱和脂肪酸有反应，但对二十碳五烯酸、花生四烯酸和二十二碳六烯酸没有反应。由于ACS- ii能轻微激活饱和脂肪酸，因此可以用ACS- ii和细菌ACS联合测定二十碳五烯酸、花生四烯酸和二十二碳六烯酸。

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.