| 18834848 |
Seethala R, Peterson T, Dong J, Chu CH, Chen L, Golla R, Ma Z, Panemangalore R, Lawrence RM, Cheng D: A simple homogeneous scintillation proximity assay for acyl-coenzyme A:diacylglycerol acyltransferase. Anal Biochem. 2008 Dec 15;383(2):144-50. Epub 2008 Sep 15.
Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is a key enzyme in triacylglycerol synthesis, and inhibiting this enzyme is a promising approach for treating obesity, type II diabetes, and dyslipidemia. There are two distinct DGAT enzymes: DGAT1 and DGAT2. The conventional assay for measuring DGAT activity is a thin layer chromatography (TLC) method, which is not amenable to screening a large number of compounds. To increase the throughput, we have developed a novel, homogeneous scintillation proximity assay (SPA) for DGAT. In this assay, when (3) H-labeled acyl-CoA is used as the acyl donor and diacylglycerol is used as the acyl acceptor, the (3) H-labeled triacylglycerol product formed in the reaction binds to polylysine SPA beads, producing a signal that is measured in a TopCount or LEADseeker. The apparent Michaelis-Menten kinetic parameters determined by this DGAT SPA method agreed well with the values determined with the conventional TLC assay. The statistical values also indicate that the DGAT SPA is a robust assay, with a Z' of more than 0.60 and a signal/background ratio of approximately 9. These results suggest that the current assay provides high-throughput capacity for the identification of DGAT inhibitors. |
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