Z pro prolinal

ACE2 enzymatic activity assay was performed as previously published by our group [25 (link),44 (link),45 (link),46 (link)] and adapted to different tissues. Briefly, 5 μL of serum or 5 μg of tissue samples that were previously homogenized were incubated using a 100 mM Tris-HCl, 600 mM NaCl, 10 μM ZnCl₂, pH 7.5 buffer in the presence of protease inhibitors containing 100 μM captopril, 5 μM amastatin, 5 μM bestatin (all from Sigma-Aldrich, Madrid, Spain), and 10 μM Z-Pro-prolinal (Enzo Life Sciences, Grupo Taper, Madrid, Spain). Samples were incubated with 20 μM Mca-Ala-Pro-Lys(Dnp)-OH (Enzo Life Sciences), a specific ACE2 quenched fluorogenic substrate, at 37 °C. Enzymatic activity was determined after 4 hours of incubation in tissue, and 16 h of incubation in serum. The plates were read using a fluorescence plate reader Tecan Infinite 200 (TECAN Instruments) at an excitation wavelength of 320 nm and an emission wavelength of 400 nm. Results were expressed as RFU (Relative Fluorescent Units) per μL of sample or μg of protein and per hour (RFU/μl/h or RFU/μg/h).

Kidney cortex samples were homogenized in a buffer consisting of 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 0.025 mM ZnCl₂, and 0.1 mM Pefabloc SC Plus (Roche) and EDTA-free protease inhibitor cocktail tablet (Roche) and clarified by centrifugation at 14,000×g for 10 min at 4°C. After measuring protein concentration by Micro BCA assay kit (Thermo Scientific), tissue samples were diluted in a buffer containing 100 mM Tris-HCl, 600 mM NaCl, 10 µM ZnCl2, pH 7.5 and 100 µM captopril, 5 µM amastatin, 5 µM bestatin (all from Sigma-Aldrich), and 10 µM Z-Pro-prolinal (Enzo Life Sciences). To each well, 40 µl of a diluted tissue sample (0.5 µg of total renal protein) was added, along with 10 µl of buffer (with or without an specific ACE2 inhibitor, MLN-4760), and the reaction was initiated by the addition of 50 µl of the substrate (5 µmol/l, final concentration). Kidney cortex ACE2 activity was determined after 4-hour incubation at 37°C. The plates were read as described above. Experiments were carried out in duplicate for each data point. Results after subtraction of the inhibition value were expressed as RFU (Relative Fluorescent Units) per µg of protein and per hour (RFU/µg/hr).

The ACE2 fluorescent enzymatic assay protocol was performed as previously described with modifications, using a specific ACE2 quenched fluorogenic substrate (Mca-Ala-Pro-Lys(Dnp)-OH, Enzo Life Sciences) [26] (link), [27] (link). Briefly, 5 µL of serum or 2 µL of diluted urine (1∶10) samples were incubated with a buffer (100 mM Tris-HCl, 600 mM NaCl, 10 µM ZnCl2, pH 7.5) in the presence of protease inhibitors, including 100 µM captopril, 5 µM amastatin, 5 µM bestatin (all from Sigma-Aldrich), and 10 µM Z-Pro-prolinal (Enzo Life Sciences). Samples were incubated with 20 µM quenched fluorogenic substrate in reaction buffer (final reaction volume 100 µL) at 37°C. Serum and urine ACE2 activity was determined after 16 hours of incubation. The plates were read using a fluorescence plate reader Tecan Infinite 200 (TECAN Instruments) at an excitation wavelength of 320 nm and an emission wavelength of 400 nm. Results were expressed as RFU (Relative Fluorescent Units) per µl of sample and per hour (RFU/µl/hr).