反应详情

For enzyme assay, 100-200 μl of enzyme was incubated in the presence and absence of test compound (prepared in DMSO) for 10 minutes at 30° C. in 0.05M Tris-HCl buffer (pH 7.2) containing 1 mM glutathione, 2 mM CaCl2, 14 μM indomethacin and 0.25 and 0.5 mM EDTA (final volume 400 μl). The final DMSO concentration in these assays was 4%. The assay was initiated by the addition of 100 μl 14C-arachidonic acid (58 mCi/mmole) and incubations were carried out for 10 minutes at 37° C. in a shaking water bath. The reactions were stopped by the simultaneous addition of 4.0 ml of diethylether and 100 μl of 1.0 m citric acid, while the tubes were vigorously vortexed. The 14C-5-HETE generated by the enzyme was extracted into the acidified ether layer which was separated from the aqueous layer by freezing the assay tubes in a dry ice/acetone bath prior to decanting the liquid ether layer. The ether layer was evaporated to dryness under N2, the residue dissolved in 65 μl chloroform:methanol (2:1 v/v), prior to separating 14C-5-HETE from other 14C-labeled compounds on silica gel impregnated glass fiber TLC plates utilizing an elution solvent of isoctane:methylethylketone:acetic acid (100:9:1 v/v). 14C-5-HETE was identified by its co-chromatography with a chemically synthesized standard. The radioactive peaks of the chromatogram were cut out and the radioactivity was quantitated by liquid scintillation counting. The amount of 5-HETE obtained per sample was expressed as the percent of total and was calculated in the basis of: ##EQU2## in order to evaluate the effect of the test compounds, the mean percent inhibition of 14C-5-HETE production was calculated. The IC50 for a drug was determined from a linear regression analysis of a plot of the mean percent inhibition vs drug concentration.

1. concentrationThe final DMSO concentration in these
2. additionThe assay was initiated by the addition of 100 μl 14C-arachidonic acid (58 mCi/mmole) and incubations
3. additionThe reactions were stopped by the simultaneous addition of 4.0 ml of diethylether and 100 μl of 1.0 m citric acid, while the tubes
4. extractionwas extracted into the acidified ether layer which
5. customwas separated from the aqueous layer
6. customto decanting the liquid ether layer
7. customThe ether layer was evaporated to dryness under N2
8. dissolutionthe residue dissolved in 65 μl chloroform:methanol (2:1 v/v)
9. customto separating 14C-5-HETE from other 14C-