Octaethylene glycol monododecyl ether

Kinetic parameters (K_M and k_cat) of N‐acetyl‐l‐aspartyl‐l‐glutamate (NAAG) cleavage by Avi‐mGCPII were determined as previously described 34, with a minor modification: The reactions were performed in a 96‐well plate, and appropriate amounts of Avi‐mGCPII were mixed with 25 mm Bis‐Tris propane, 150 mm NaCl, 0.001% octaethylene glycol monododecyl ether (Affymetrix, Santa Clara, CA, USA), pH 7.4.

A small piece of tissue (approx. 30 mg) was transferred into 250 μL of 50 mm Tris/HCl, 100 mm NaCl, pH 7.4, in a 2‐mL microtube. Tissue samples were homogenized using TissueLyser II (30 Hz, 3 min). The homogenates were then diluted with 250 μL of the lysis buffer. Octaethylene glycol monododecyl ether (Affymetrix) was added to reach 1% final concentration, and the homogenate was sonicated in a water bath for 5 min at 0 °C. Finally, the samples were centrifuged at 600 g for 15 min, and the resulting supernatant was stored at −80 °C until further use. The lysate protein concentration was determined using Bradford 1 × Dye Reagent (Bio‐Rad, Hercules, CA, USA).

Kinetic parameters (K_M and k_cat) of pteroyl‐di‐l‐glutamate cleavage by Avi‐mGCPII, as well as K_i values for all inhibitors, were determined as previously described 39. Briefly, in a 96‐well plate, Avi‐mGCPII was mixed with 25 mm Bis‐Tris propane, 150 mm NaCl, 0.001% octaethylene glycol monododecyl ether (Affymetrix), pH 7.4 (and tested inhibitor, if used), into a final volume of 90 μL. Reactions were started by adding 10 μL of 4 μm pteroyl‐di‐l‐glutamate and incubated at 37 °C for 20 min. The reactions were stopped with 20 μL of 25 μm 2‐PMPA and subsequently analyzed on an Agilent 1200 Series system using an Acquity UPLC HSS T3 1.8 μm column (2.1 × 100 mm; Waters, Milford, MA, USA).