Z gly pro amc

For the preparation of tissue homogenates, 20 mg of N₂‐pulverized rat brain were suspended in 250 μL PREP lysis buffer (0.1 mol/L Tris‐HCl pH 7.4, 1 mmol/L EDTA, 1% octyl glucoside, 50 μg/mL aprotinin, 5 mmol/L DTT). The samples were left on ice for 1 hour and centrifuged at 4°C at 12 000 g for 15 minutes. PREP activities were measured kinetically on the clarified supernatants using Z‐Gly‐Pro‐pNA (Bachem, Bubendorf, Switzerland) (250 μmol/L, 5% dimethyl sulfoxide [DMSO]) in 0.1 mol/L Tris‐HCl pH 7.4, 1 mmol/L EDTA, 3 mM DTT as described.38For determining PREP activity in rat plasma, the fluorescent substrate Z‐Gly‐Pro‐AMC (Bachem, Bubendorf, Switzerland; 250 μmol/L in 5% DMSO) was used. The activities were determined kinetically for 60 minutes at 37°C by measuring the velocity of AMC release (λ_ex = 380 nm, λ_em = 460 nm). Fluorescence intensity was related to an AMC standard curve in the same buffer. One unit of activity was defined as the amount of enzyme catalyzing the conversion of 1 μmol of substrate in 1 minute under reaction conditions. All activities were normalized to the protein content in the lysates as determined in a Bradford assay.

Human recombinant His-tagged PREP and KYP-2047 (UAMC-714) were kindly provided by Dr. Anne-Marie Lambeir (University of Antwerp, Belgium). Purified PREP was incubated with DMSO or a serine hydrolase inhibitor for 15 min at 37°C. Subsequently, 200 μM of z-Gly-Pro-AMC (Bachem), a fluorogenic substrate of PREP, was added. The reaction was performed in 50 mM phosphate buffer (pH 8) supplemented with 1 mM DTT. Fluorescence was measured with a FLUOstar Galaxy microplate reader (BMG Labtech) at excitation and emission wavelengths of 390 and 460 nm respectively.