反应详情

Intrinsic activity of HMG-CoA synthase is measured in the following in vitro assay. Enzyme protein (ca. 12.2 μg) is added to a solution containing 117 mM Tris-HCl (pH 8.0), 11.7 mM MgCl2, 1.17 mM Ethylenediaminetetraacetic acid (EDTA), 0.58 mM dithiothreitol, in the presence or absence of the test compound (added as, e.g., a 2 μg/ml solution in dimethylsulfoxide). The incubation is in a volume of 0.085 ml at 30° C. in a shaking water bath. After 5 minutes, 15 μl of a solution containing acetoacetyl-CoA and 0.1 μCi of 1[14C]-acetyl-CoA is added to give final concentrations of 0.1 and 0.4 mM, respectively. The incubation is continued for 2 more minutes and the reaction stopped by the addition of 50 μl of the assay mixture to 0.2 ml of 6N HCl in a glass scintillation vial. The vial is heated for 1 hour at 110° C. after which time 0.2 ml more of 6N HCl is again added to each vial and the heating continued for another hour. Following this, 1.0 ml of 0.9% saline is added to each vial and finally 10 ml of scintillation liquid. Radioactivity is determined in a Packard Tri-Carb liquid scintillation counter. Percent inhibition is calculated a standard formula. IC50 values are determined by plotting the log of the concentration of the test compound verses the percentage inhibition and fitting a straight line to the resulting data by using the least squares method.

1. additionEnzyme protein (ca. 12.2 μg) is added to a solution
2. additionin the presence or absence of the test compound (added as, e.g.
3. customThe incubation is in a volume of 0.085 ml at 30° C. in a shaking water bath
4. additionis added
5. customto give final concentrations of 0.1 and 0.4 mM
6. additionis again added to each vial and the heating
7. waitcontinued for another hour
8. additionis added to each vial and finally 10 ml of scintillation liquid