Enzyme Assay: Primary stock solutions of test compounds at a concentration of 6 mM were prepared from the 2 to 5 mg powder. The primary stock solutions of each test compound were prepared freshly in distilled water on the day of study to obtain a final concentration of 6 mM. For determination of IC50 values, 12 test compound concent rations were prepared as 3-fold serial dilutions. Concentration range of test compound utilized for nNOS were 0.001 to 300 μM and for eNOS were 0.003 to 10 00 μM. The vehicle of the test compound or inhibitor was used as blank control. For non-specific activity, 100 μM L-NMMA was used. Runs using the IC50 concentration of L-NAME were done in parallel as controls. All incubations are performed in duplicate.
Enzyme Assay: Recombinant human inducible NOS (iNOS) was produced in Baculovirus-infected Sf9 cells (ALEXIS). In a radiometric method, NO synthase activity was determined by measuring the conversion of [3H]L-arginine to [3H]L-citrulline. To measure iNOS, 10 uL of enzyme was added to 100 uL of 100 mM HEPES, pH=7.4, containing 1 mM CaCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 uM FMN, 1 uM FAD, 10 uM tetrahydrobiopterin, 120 uM NADPH, and 100 nM CaM. To measure enzyme inhibition, a 15 uL solution of a test substance was added to the enzyme assay solution, followed by a pre-incubation time of 15 min at RT. The reaction was initiated by addition of 20 uL L-arginine containing 0.25 uCi of [3H] arginine/mL and 24 uM L-arginine. The total volume of the reaction mixture was 150 uL in every well.
Enzyme Assay: Primary stock solutions of test compounds at a concentration of 6 mM were prepared from the 2 to 5 mg powder. The primary stock solutions of each test compound were prepared freshly in distilled water on the day of study to obtain a final concentration of 6 mM. For determination of IC50 values, 12 test compound concent rations were prepared as 3-fold serial dilutions. Concentration range of test compound utilized for nNOS were 0.001 to 300 μM and for eNOS were 0.003 to 10 00 μM. The vehicle of the test compound or inhibitor was used as blank control. For non-specific activity, 100 μM L-NMMA was used. Runs using the IC50 concentration of L-NAME were done in parallel as controls. All incubations are performed in duplicate.