High-performance liquid chromatographic assay with ultraviolet spectrometric detection for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C

A nonradioactive spectrometric assay for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C (PI–PLC) is described. l-α-Phosphatidylinositol from bovine liver was used as substrate in the presence of the micelle-forming detergent deoxycholic acid. PI–PLC isolated from Bacillus cereus and crude cytosol fractions from porcine brain were used as enzyme sources. PI–PLC activity was determined by measuring the release of 1-stearoyl-2-arachidonoyl-sn-glycerol with reversed-phase HPLC and UV detection at 200 nm. PI–PLC from B. cereus was not inhibited by the putative PI–PLC inhibitors U-73122 and ET-18-OCH3 at 100 μM, whereas the isobenzofuranone derivative 5 blocked the enzyme with an IC50 of 75 μM. PI–PLC activity present in porcine brain cytosol was decreased by all three test compounds at 100 μM to approximately 30 to 50%.