title: Measurement of Long-Chain Fatty Acyl-CoA Synthetase Activity.
abstract: Long-chain fatty acyl-CoA synthetases (ACS) are a family of essential enzymes of lipid metabolism, activating fatty acids by thioesterification with coenzyme A. Fatty acyl-CoA molecules are then readily utilized for the biosynthesis of storage and membrane lipids, or for the generation of energy by ß-oxidation. Acyl-CoAs also function as transcriptional activators, allosteric inhibitors, or precursors for inflammatory mediators. Recent work suggests that ACS enzymes may drive cellular fatty acid uptake by metabolic trapping, and may also regulate the channeling of fatty acids towards specific metabolic pathways. The implication of ACS enzymes in widespread lipid associated diseases like type 2 diabetes has rekindled interest in this protein family. Here, we describe in detail how to measure long-chain fatty acyl-CoA synthetase activity by a straightforward radiometric assay. Cell lysates are incubated with ATP, coenzyme A, Mg(2+), and radiolabeled fatty acid bound to BSA. Differential phase partitioning of fatty acids and acyl-CoAs is exploited to quantify the amount of generated acyl-CoA by scintillation counting. The high sensitivity of this assay also allows the analysis of small samples like patient biopsies.
authors.affiliation: Molecular Cell Biology Laboratory Internal Medicine IV, Heidelberg University Hospital, Otto-Meyerhof-Zentrum, University of Heidelberg, Im Neuenheimer Feld 350, 69120, Heidelberg, Germany. Joachim.Fuellekrug@med.uni-heidelberg.de.
authors.forename: Joachim
authors.initials: J
authors.lastname: Füllekrug
authors.affiliation: Molecular Cell Biology Laboratory Internal Medicine IV, Heidelberg University Hospital, Otto-Meyerhof-Zentrum, University of Heidelberg, Im Neuenheimer Feld 350, 69120, Heidelberg, Germany.
authors.forename: Margarete
authors.initials: M
authors.lastname: Poppelreuther
mesh_terms: D000214:Acyl Coenzyme A / Q000096:biosynthesis*; D000818:Animals; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D012588:Scintillation Counting / Q000379:methods
chemical_list: D000214:Acyl Coenzyme A; D005227:Fatty Acids; D003066:Coenzyme A Ligases
keywords: Acyl-CoA synthetase; Fatty acid; Fatty acid CoA ligase; Lipid metabolism; Oleic acid; Thioesterification