MCA-APK(DNP) IS NOT A SELECTIVE SUBSTRATE OF ANGIOTENSIN-CONVERTING ENZYME-2

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Objective. This study was conducted to ascertain the specificity of MCA-APK(Dnp) for angiotensin-converting enzyme2 (ACE-2). Background. MCA-APK(Dnp) is a fluorogenic substrate used in assays of ACE-2 metabolism. Cleavage of the Pro-Lys bond splits off the quenching Dnp group, allowing the MCA to fluoresce. Methods. Frozen rat tissues were homogenized in 19 volumes of 50 mM sodium phosphate at pH 7 with 0.05% Triton X-100 detergent and centrifuged at 48,000 x g. The supernatant was then diluted 20-fold in assay buffer for a final concentration of 50 mM sodium phosphate at pH 6, 6.5, 7, or 7.5 and 100 mM NaCl. Substrate metabolism was measured in the presence or absence of the ACE-2 inhibitor MLN-4760. 20 μL of diluted supernatant, 10 μl of 50 μM MLN-4760, and 20 μl of 125 μM substrate were added to each well for a reaction volume of 50 μL. Metabolism of MCA-APK(Dnp) was recorded at 37°C at a wavelength of 393 nm with excitation at 328 nm. Results. Breakdown of MCA-APK(Dnp) attributable to ACE-2 represented only 21%, 28%, 44%, and 56% of total enzymatic activity at pH 6, 6.5, 7, and 7.5, respectively. Total enzymatic activity also increased with decreasing pH. Conclusion. Our results suggest that MCA-APK(Dnp) is not selective for ACE-2. When using MCA-APK(Dnp) as a surrogate substrate for determination of ACE-2 activity, it is critical to use a selective inhibitor of ACE-2 activity such as MLN-4760 to differentiate ACE-2 versus non-ACE-2 mediated metabolism. Grants. This study was funded by NIH-NHLBI HL113905.