Journal: Pharmacology Research & Perspectives

Article Title: Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917

doi: 10.1002/prp2.138

Figure Lengend Snippet: Purification and identification of CS-917 esterase activity from the monkey liver. (A) The CS-917 esterase activity precipitated by ammonium sulfate from monkey liver was subjected to a hydrophobic interaction (HIC). The activity was tested in the absence (gray bars) or presence (hatched bars) of 1 mmol/L BNPP, a CES specific inhibitor. CS-917 esterase activity of each active peak in the HIC was separately purified by successive chromatography. The CS-917 esterase activities were separated by the final anion exchange column purified from the first (B) or the second (D) active peak. The BNPP inhibitory effect was further tested on the active fractions from the first (C) or the second (E) active peak (gray and hatched bars). Quantitation of CES1 (C) and CTSA (E) by mass spectrometry using emPAI (closed and open box lines) correlated well with the enzyme activity. Four independent experiments were conducted, and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. CTSA, cathepsin A; BNPP, bis(4-nitrophenyl) phosphate.

Article Snippet: The active fractions (3 mL) were dialyzed against AEX buffer (20 mmol/L Tris, pH 10.0, containing 0.1% CHAPS, 5 mmol/L MgCl 2 , and loaded onto an anion exchange column (Mini Q PC 3.2/3; GE Healthcare).

Techniques: Purification, Activity Assay, Chromatography, Quantitation Assay, Mass Spectrometry