Detection of Acyl-CoA Derivatized with Butylamide for in vitro Fatty Acid Desaturase Assay

Abstract

Membrane-bound fatty acid desaturases acting on acyl-CoA contribute to the biosynthesis of unsaturated fatty acids, such as arachidonic acid and docosahexaenoic acid in higher organisms. We propose a simplified method for measuring the desaturase activity that combines the in vitro reaction by desaturase-expressing yeast cell homogenate and the detection of acyl-CoA product as butylamide derivatives by gas chromatography. To set up the in vitro reaction, we traced the in vivo activity of rat liver ∆6 fatty acid desaturase (D6d) expressed in the yeast, Saccharomyces cerevisiae, and determined the time taken for the D6d activity to reach its maximum level. The cell homogenate of yeast expressing the maximum D6d activity was made to react in vitro with linoleoyl-CoA to generate the D6d product, γlinolenoyl-CoA. This product was successfully detected as a peak corresponding to γ-linolenoyl butylamide on gas chromatography. This procedure, with low background expression, using non-labeled acyl-CoA as substrate, will contribute toward developing a simple in vitro desaturase assay. It will also help in elucidating the functions of membrane-bound fatty acid desaturases with various substrate specificities and regioselectivities.