Measurement of Long-Chain Fatty Acyl-CoA Synthetase Activity
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, Mg2+, 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.
Key wordsAcyl-CoA synthetase Fatty acid Lipid metabolism Oleic acid Thioesterification Fatty acid CoA ligase
This work was supported by DFG Grants FU 340/7-1 (to J.F.) and PO 1767/3-1 (to M.P.). We gratefully acknowledge Ina Feldhoffer and Svenja Sliwinski from our lab who did the cross-reading from a student’s perspective.
- 2.Mashek DG, Bornfeldt KE, Coleman RA, Berger J, Bernlohr DA, Black P, DiRusso CC, Farber SA, Guo W, Hashimoto N, Khodiyar V, Kuypers FA, Maltais LJ, Nebert DW, Renieri A, Schaffer JE, Stahl A, Watkins PA, Vasiliou V, Yamamoto TT (2004) Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family. J Lipid Res 45(10):1958–1961PubMedCrossRefGoogle Scholar