Prolyl oligopeptidase activity assay.

Meprin-α activity was measured with a fluorogenic substrate, Mca-YVADAPK(Dnp)-OH (19). Kidney homogenates (50 μg) from Sprague-Dawley rats were incubated for 30 min at 37°C and pH 7.4 with 10 μmol/l fluorogenic substrate in PBS containing the following broad-range protease inhibitors: cOmplete EDTA-free cocktail from Roche, 1 μg/ml pepstatin A, 10 μmol/l leupeptin, 1 mmol/l CaCl₂, and 1 mmol/l ZnCl₂. The fluorescence of the released product was measured using a fluorometer (SpectraMax M2) at an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Relative meprin-α activity is expressed in arbitrary fluorescence units per minute per milligram of total protein. The meprin-α inhibitor actinonin (100 μmol/l) was used as a control. POP enzyme activity was measured as described previously using a fluorogenic substrate, Z-Gly-Pro-AMC (19). Briefly, kidney homogenates from the cortex and the medulla were obtained using a cold Stadie-Riggs microtome. Protein homogenates (50 µg) were incubated with 50 µmol/l fluorogenic substrate in PBS, pH 7.4, for 60 min at 37°C. The fluorescence of the released 7-amino-4-methylcoumarin product was measured at an excitation of 360 nm and an emission of 460 nm and was quantified against AMC standards. Specific POP activity is expressed as picomoles AMC per minute per milligram total protein. A POP inhibitor, {"type":"entrez-protein","attrs":{"text":"S17092","term_id":"94591","term_text":"pir||S17092"}}S17092 (20 µmol/l), was used as a control.