Measurement of Long-Chain Fatty Acyl-CoA Synthetase Activity

  • Joachim FüllekrugEmail author
  • Margarete Poppelreuther
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)


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 words

Acyl-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.


  1. 1.
    Watkins PA, Maiguel D, Jia Z, Pevsner J (2007) Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome. J Lipid Res 48(12):2736–2750PubMedCrossRefGoogle Scholar
  2. 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
  3. 3.
    Watkins PA (2008) Very-long-chain acyl-CoA synthetases. J Biol Chem 283(4):1773–1777. doi: 10.1074/jbc.R700037200 PubMedCrossRefGoogle Scholar
  4. 4.
    Pérez-Chacón G, Astudillo AM, Balgoma D, Balboa MA, Balsinde J (2009) Control of free arachidonic acid levels by phospholipases A2 and lysophospholipid acyltransferases. Biochim Biophys Acta 1791(12):1103–1113PubMedCrossRefGoogle Scholar
  5. 5.
    Grevengoed TJ, Klett EL, Coleman RA (2014) Acyl-CoA metabolism and partitioning. Annu Rev Nutr. doi: 10.1146/annurev-nutr-071813-105541 PubMedGoogle Scholar
  6. 6.
    Soupene E, Kuypers FA (2008) Mammalian long-chain acyl-CoA synthetases. Exp Biol Med 233(5):507–521CrossRefGoogle Scholar
  7. 7.
    Li LO, Klett EL, Coleman RA (2009) Acyl-CoA synthesis, lipid metabolism and lipotoxicity. Biochim Biophys Acta 1801(3):246–251PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Faergeman NJ, Knudsen J (1997) Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling. Biochem J 323(Pt 1):1–12PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Golej DL, Askari B, Kramer F, Barnhart S, Vivekanandan-Giri A, Pennathur S, Bornfeldt KE (2011) Long-chain acyl-CoA synthetase 4 modulates prostaglandin E2 release from human arterial smooth muscle cells. J Lipid Res 52(4):782–793. doi: 10.1194/jlr.M013292 PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Mashek DG, Li LO, Coleman RA (2006) Rat long-chain acyl-CoA synthetase mRNA, protein, and activity vary in tissue distribution and in response to diet. J Lipid Res 47(9):2004–2010PubMedCrossRefGoogle Scholar
  11. 11.
    Digel M, Staffer S, Ehehalt F, Stremmel W, Ehehalt R, Füllekrug J (2011) FATP4 contributes as an enzyme to the basal and insulin-mediated fatty acid uptake of C2C12 muscle cells. Am J Physiol Endocrinol Metab 301(5):E785–E796PubMedCrossRefGoogle Scholar
  12. 12.
    Smith ME, Saraceno GE, Capani F, Castilla R (2013) Long-chain acyl-CoA synthetase 4 is regulated by phosphorylation. Biochem Biophys Res Commun 430(1):272–277. doi: 10.1016/j.bbrc.2012.10.138, Scholar
  13. 13.
    Kampf JP, Kleinfeld AM (2007) Is membrane transport of FFA mediated by lipid, protein, or both? Physiology (Bethesda) 22:7–14CrossRefGoogle Scholar
  14. 14.
    Milger K, Herrmann T, Becker C, Gotthardt D, Zickwolf J, Ehehalt R, Watkins PA, Stremmel W, Füllekrug J (2006) Cellular uptake of fatty acids driven by the ER-localized acyl-CoA synthetase FATP4. J Cell Sci 119(Pt 22):4678–4688PubMedCrossRefGoogle Scholar
  15. 15.
    Black PN, DiRusso CC (2003) Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification. Microbiol Mol Biol Rev 67(3):454–472PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Füllekrug J, Ehehalt R, Poppelreuther M (2012) Outlook: membrane junctions enable the metabolic trapping of fatty acids by intracellular acyl-CoA synthetases. Front Physiol 3:401. doi: 10.3389/fphys.2012.00401 PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Digel M, Ehehalt R, Stremmel W, Füllekrug J (2009) Acyl-CoA synthetases: fatty acid uptake and metabolic channeling. Mol Cell Biochem 326(1-2):23–28. doi: 10.1007/s11010-008-0003-3 PubMedCrossRefGoogle Scholar
  18. 18.
    Kim J-H, Lewin TM, Coleman RA (2001) Expression and characterization of recombinant rat acyl-CoA synthetases 1, 4, and 5. Selective inhibition by triacsin C and thiazolidinediones. J Biol Chem 276(27):24667–24673PubMedCrossRefGoogle Scholar
  19. 19.
    Hall AM, Smith AJ, Bernlohr DA (2003) Characterization of the acyl-CoA synthetase activity of purified murine fatty acid transport protein 1. J Biol Chem 278(44):43008–43013PubMedCrossRefGoogle Scholar
  20. 20.
    Coe NR, Smith AJ, Frohnert BI, Watkins PA, Bernlohr DA (1999) The fatty acid transport protein (FATP1) is a very long chain acyl-CoA synthetase. J Biol Chem 274(51):36300–36304PubMedCrossRefGoogle Scholar
  21. 21.
    Uchida Y, Kondo N, Orii T, Hashimoto T (1996) Purification and properties of rat liver peroxisomal very-long-chain acyl-CoA synthetase. J Biochem (Tokyo) 119(3):565–571CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Molecular Cell Biology Laboratory Internal Medicine IV, Heidelberg University Hospital, Otto-Meyerhof-ZentrumUniversity of HeidelbergHeidelbergGermany

Personalised recommendations