Figure 5. pH optima of mouse (m) and human (h) BAT5 hydrolase activities towards 1-LG and 15d-PGJ₂-G.

Lysates (0.3 µg/well) of HEK293 cells transiently expressing mBAT5 (B, E) or hBAT5 (C, F) were incubated at the indicated pH for 60 min using 1-LG (A, B, C) or 15d-PGJ₂-G (D, E, F) as the substrate (25 µM final concentration). For comparative purposes, substrate hydrolysis in lysates of parental HEK293 cells is also illustrated (A, D). The incubations additionally contained 0.1% (v/v) ethanol, 0.1% (v/v) DMSO and 0.5% (w/v) BSA. After 60 min incubation, the pH of the assay system was brought to the neutral range by the addition of Glycerol Assay Mix containing additionally 100 mM Tris-HCl, pH 7.4 and THL (10⁻⁵ M) to quench hydrolase activity. Assay blanks and glycerol standards were included for each pH condition. Glycerol content was determined at time-point 60 min. The buffer systems were as follows: 10 mM K-phosphate buffer (KPB) covering the pH range 5.3–8.0 and 10 mM Tris-HCl covering the pH range 7.2–9.1. For comparative purposes, hydrolase activity in the routinely used hydrolase assay buffer (TEMN, pH 7.4) is shown. Data are mean + SEM of duplicate wells from two independent experiments.