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-alpha-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-OCH(3) at 100 microM, whereas the isobenzofuranone derivative 5 blocked the enzyme with an IC(50) of 75 microM. PI-PLC activity present in porcine brain cytosol was decreased by all three test compounds at 100 microM to approximately 30 to 50%.
Schmolke
Marion
M
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, D-48149 Münster, Germany.
Fabian
Jörg
J
Lehr
Matthias
M
D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology; D001921:Brain / Q000166:cytology / Q000201:enzymology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
C011439:1,2-diacylglycerol; D004075:Diglycerides; D004791:Enzyme Inhibitors; D010716:Phosphatidylinositols; C075809:1-stearoyl-2-arachidonoylglycerol; D054801:Phosphoinositide Phospholipase C