| Assay Method Information | |
| | Enzyme Activity Assay |
| Description: | Recombinant ECE2 (32.5 ng) with a specific activity of 12 pmol/min/μg protein was generated as described previously. Secreted soluble recombinant ECE1 (30 ng) with a specific activity of 750 pmol/min/μg protein was generated and purified using a protocol similar to that used for ECE2. Solubilized midbrain membranes (10 μg) from wildtype or ECE2 knock-out mice were prepared as described. Enzymatic activity, in the absence or presence of the ECE2 inhibitor S136492 or the ECE1 inhibitor SM19712, was assayed using the synthetic quenched fluorescent substrate McaBk2 (10 μM) at 37 °C with either 0.2 M sodium acetate buffer, pH 5.5, or 50 mM Tris-Cl buffer, pH 7.4, as described previously [Gagnidze et al., J. Med. Chem., 51:3378-3387; Ouimet et al., J. Biol. Chem., 285:34390-34400]. |
| Affinity data for this assay | |
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