A homogeneous scintillation proximity assay for acetyl coenzyme A carboxylase coupled to fatty acid synthase.
We have devised a rapid and sensitive homogeneous assay for acetyl CoA carboxylase (ACC) in a scintillation proximity assay format suitable for high-throughput screening. In this assay, ACC is coupled to fatty acid synthase (FAS). Malonyl CoA, the product of the ACC reaction, and acetyl CoA serve as substrates for FAS to synthesize palmitic acid. When [(3)H]acetyl CoA is used in the ACC/FAS coupled system, [(3)H]palmitic acid, the final product, is readily detected by scintillation proximity in a FlashPlate or Image FlashPlate coated with phospholipid. The [(3)H]palmitic acid binds to the coated phospholipid through hydrophobic interaction which brings it into close proximity of the scintillant on the FlashPlate or the Image FlashPlate, yielding photons that are read in a TopCount or LeadSeeker, respectively. The current assay consists of simple reagent addition, incubation, and detection of signal. The signal is approximately 30-fold over the background and the Z' value is approximately 0.80, suggesting that this assay is robust and highly reproducible. To our knowledge this ACC/FAS coupled scintillation proximity assay is the only assay format that is compatible with high-throughput screening for systematic search of inhibitors against mammalian ACC.
Seethala
Ramakrishna
R
Division of Drug Discovery, Pharmaceutical Research Institute, Bristol-Myers Squibb Co., P.O. Box 5400, Princeton, NJ 08543-5400, USA. ramakrishna.seethala@bms.com <ramakrishna.seethala@bms.com>
Ma
Zhengping
Z
Golla
Rajasree
R
Cheng
Dong
D
D000103:Acetyl-CoA Carboxylase / Q000378:metabolism*; D000818:Animals; D064429:Fatty Acid Synthases / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012588:Scintillation Counting / Q000379:methods*
D064429:Fatty Acid Synthases; D000103:Acetyl-CoA Carboxylase