反应详情

4-Methylumbelliferyl sulfate (final concentration 0.3 mmol/L), recombinant human estrone sulfatase (7.8 ng/well) and an estra-1,3,5(10)-triene derivative of each test concentration (the estra-1,3,5(10)-triene derivative was dissolved in 0.001 mL of DMSO) were added to a phosphate buffer (pH 7.5, final volume 0.1 mL) containing 0.28 mol/L of sucrose and 0.04 mol/L of nicotinamide, and the mixture was subjected to enzymatic reaction at 37° C. for 1 hour. The recombinant human estrone sulfatase used herein was prepared from CHO (Chinese hamster ovary) cells, which were transfected with a human estrone sulfatase gene, after partial purification. After the enzymatic reaction, an aqueous solution of 2 mol/L glycine-sodium hydroxide (pH 10.3, 0.13 mL) was added thereto followed by stirring. Using a fluorescent plate reader (Cyto Fluor II), the fluorescent intensity of 4-methylumbelliferone formed in the system was measured. The measurement was carried out at one time by duplication, and the assay fluctuation was 10% or less. The enzymatic reaction in the absence of the test compound was also carried out simultaneously. The inhibitory activity against estrone sulfatase of the test compound of each concentration was calculated according to the equation shown below. Inhibitory Activity against Estrone Sulfatase (%)=100−[100×(A/B)] wherein;

1. concentration4-Methylumbelliferyl sulfate (final concentration 0.3 mmol/L), recombinant human estrone sulfatase (7.8 ng/well) and an estra-1,3,5(10)-triene derivative of each test concentration (the estra-1,3,5(10)-triene derivative
2. dissolutionwas dissolved in 0.001 mL of DMSO)
3. additionwere added to a phosphate buffer (pH 7.5
4. additionfinal volume 0.1 mL) containing 0.28 mol/L of sucrose and 0.04 mol/L of nicotinamide
5. customthe mixture was subjected to enzymatic reaction at 37° C. for 1 hour
6. customwas prepared from CHO (Chinese hamster ovary) cells, which
7. customafter partial purification
8. customAfter the enzymatic reaction