A homogeneous scintillation proximity format for monitoring the activity of recombinant human long-chain-fatty-acyl-CoA synthetase 5.
Fatty acyl coenzyme A (CoA) synthetases are a group of enzymes responsible for the activation of fatty acids through ligated high-energy CoA thioester bonds. Ultimately these fatty acyl-CoA conjugates are routed toward either anabolic or catabolic pathways. Long-chain-fatty-acid-CoA ligase 5 (LACS 5) utilizes a wide range of saturated fatty acids with a substrate preference for C16-C18 unsaturated fatty acids. This enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a radiometric homogeneous measurement of the enzymatic reaction by employing ionic capture of the reaction product onto YSi scintillation proximity assay (SPA) beads. We present kinetic and inhibition data for LACS 5 using this SPA format. Our results show that the assay method is both robust and well suited for this class of lipid-metabolizing enzymes.
Bembenek
Michael E
ME
Millennium Pharmaceuticals, Inc., 270 Albany Street, Cambridge, MA 02139, USA. bembenek@mpi.com
Roy
Rebecca
R
Li
Ping
P
Chee
Linda
L
Jain
Sadhana
S
Parsons
Thomas
T
D000106:Acetate-CoA Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000032:analysis / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012588:Scintillation Counting; D013379:Substrate Specificity; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D000214:Acyl Coenzyme A; D007527:Isoenzymes; D011994:Recombinant Proteins; D003066:Coenzyme A Ligases; D000106:Acetate-CoA Ligase; D000098859:Long-Chain-Fatty-Acid-CoA Ligase