تفاعل #659877

معادلة التفاعل

HCl

Tris-HCl

MgCl2

Ethylenediaminetetraacetic acid

dithiothreitol

HMG-CoA

acetoacetyl-CoA

HCl

→

CoA

المذيبات

dimethylsulfoxide

ظروف التفاعل

المعالجة

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

الإجراء التجريبي

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.

المصدر