| Assay Method Information | |
| | In Vitro APN Inhibition Assay |
| Description: | IC50 values against APN were determined as previously described and by using L-Leu-p-nitroanilide as a substrate and microsomal aminopeptidase from PorcineKidney microsomes (Sigma) in 50 mM PBS (pH 7.2) or suspension of A549, ES-2, HL-60 in PBS (2 × 10^5/well) as the enzyme. The hydrolysis of the substrate was monitored by following the change in the absorbance measured at 405 nm with a plate reader (Varioskan, Thermo, USA). All the solutions of the inhibitors wereprepared in the assay buffer, and the pH was adjusted to 7.5 by the addition of 0.1 M HCl or 0.1 M NaOH. All the inhibitors were preincubated with APN at 37°C. The assay mixture, which contained the inhibitor solution (concentration dependent on the inhibitor), the enzyme solution (4 mg/mL final concentration) or the cells suspension, and the assay buffer, was adjusted to 200 μL. |
| Affinity data for this assay | |
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