ACE and ACE2 activity assay

ACE and ACE2 activities were assayed using the fluorogenic substrates Mca-YVADAPK and Mca-APK-Dnp (AnaSpec, San Jose, CA, USA), respectively, according to the report by Vickers et al. ²⁵. The assay was performed in a microquartz cuvette with 20 μL of lung tissue proteins and 2 μL of the fluorogenic substrates (stock concentration: 4 mM ACE substrate/1.5 mM ACE2 substrate) in ACE or ACE2 assay buffer. The reaction was followed kinetically for 1 hour using a fluorescence reader at 330 nm/390 nm. All samples were fitted and plotted using Grafit v. 4.0 (Sigma-Aldrich, St. Louis, MO, USA), and enzyme activity was expressed as RFU/hour/mL. The samples were incubated with the above mentioned reaction mixture in the presence of 1 μM captopril (Sigma-Aldrich; a specific ACE inhibitor) or 1 μM DX600 (AnaSpec; a specific ACE2 inhibitor).