Quantification of ACE2 Activity in Tissue Lysates

ACE2 activity in tissue lysates was measured using specific fluorogenic ACE2 substrate (Mca-APK-(Dnp) (AnaSpec, San Jose, CA) in the presence or absence of the ACE2 inhibitor (MLN-4760) (Sigma-Aldrich, St. Louis, MO) as previously described [85 (link)]. Tissue samples were homogenized in lysis buffer (75 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.5 mM ZnCl2, 0.01 mM Captopril, 0.1 mM Z-Pro-Prolinal, 1mM PMSF, EDTA-free inhibitor cocktail tablet from Roche, and 0.5% Triton X-100) and centrifuged at 14,000 x g for 10 minutes at 4°C. Protein concentration in tissue lysates was measured using the Bradford method. Tissue lysates (10 μg of protein for kidney extracts and 40 μg of protein for heart, lung, trachea, and sinus extracts) were pre incubated with 70 μL of assay buffer (75 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.5 mM ZnCl2, 0.01 mM Captopril, 0.1 mM Z-Pro-Prolinal, and EDTA-free inhibitor cocktail tablet from Roche) with or without ACE inhibitor MLN-4760 (10 μM final) for 30 minutes at room temperature. After the incubation with ACE2 inhibitor, 30 μL of ACE2 substrate buffer (75 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.5 mM ZnCl2, 0.01 mM Captopril, 0.1 mM Z-Pro-Prolinal, and 0.167 mM Mca-APK-Dpn) was added to each well to initiate the reaction. Samples were incubated in the dark for 1 hour at room temperature, and fluorescence values were measured at an excitation wavelength of 320 nm and emission wavelength of 420 nm using a BioTek Cytation5 plate reader (BioTek instruments, Winooski, VT). Results were expressed as ΔRFU (Relative Fluorescence Unit) after subtraction of RFU values obtained in the presence of MLN-4760. BALF was collected from mice as previously described, and urine was collected during necropsy.