title: A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity.
abstract: Triacylglycerols (TG) are the major storage form of energy in eukaryotic organisms and are synthesized primarily by acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) enzymes. In vitro DGAT activity has previously been quantified by measuring the incorporation of either radiolabeled fatty acyl CoA or diacylglycerol (DG) into TG. We developed a modified acyltransferase assay using a fluorescent fatty acyl CoA substrate to accurately quantify in vitro DGAT activity. In the modified assay, radioactive fatty acyl CoA is replaced with fluorescent NBD-palmitoyl CoA, which is used as a substrate by DGAT with DG to produce NBD-TG. After extraction with organic solvents and separation by thin layer chromatography, NBD-TG formation can be detected and accurately quantified using a fluorescent imaging system. We demonstrate that this method can be adapted to detect other acyltransferase activities. Because NBD-palmitoyl CoA is commercially available at a much lower cost compared with radioactive acyl CoA substrates, it is a more economical alternative to radioactive tracers. In addition, the exposure of laboratory personnel to radioactivity is greatly reduced.
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: McFie
authors.forename: Scot J
authors.initials: SJ
authors.lastname: Stone
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D051048:Diacylglycerol O-Acyltransferase / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015394:Molecular Structure; D015203:Reproducibility of Results; D014280:Triglycerides / Q000737:chemistry / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D005456:Fluorescent Dyes; D014280:Triglycerides; D051048:Diacylglycerol O-Acyltransferase