Abstract
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 200nm. PI–PLC from B. cereus was not inhibited by the putative PI–PLC inhibitors U-73122 and ET-18-OCH3 at 100μM, whereas the isobenzofuranone derivative 5 blocked the enzyme with an IC50 of 75μM. PI–PLC activity present in porcine brain cytosol was decreased by all three test compounds at 100μM to approximately 30 to 50%.
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