MCA‐APK(Dnp) is not a selective substrate of angiotensin‐converting enzyme‐2 (1067.6)

Abstract

MCA‐APK(Dnp) is a fluorogenic substrate used in assays of angiotensin‐converting enzyme‐2 (ACE‐2) metabolism. Cleavage of the Pro‐Lys bond removes the quenching Dnp group and induces fluorescence. To ascertain the specificity of MCA‐APK(Dnp) for ACE‐2, we measured metabolism of the substrate in rat kidney in the presence or absence of the ACE‐2 inhibitor MLN‐4760. 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. 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 37oC at a wavelength of 393 nm with excitation at 328 nm. 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 activity also increased with decreasing pH. 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 or ACE‐2 activity such as MLN‐4760 to differentiate ACE‐2 versus non‐ACE‐2 mediated metabolism of MCA‐APK(Dnp).Grant Funding Source: NIH‐HL113905