Developmental Studies on Ultrasensitive Method for Determination of Polyunsaturated Fatty Acids

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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.