Ca2+ and partly GTP gamma S-dependent particulate phospholipase C hydrolyzing phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate is inhibited by diacyl(acyl-acetyl) glycerols.
The activity of a phosphodiesterase of the phospholipase C (PLC) type and factors influencing its activity were studied in ascites tumor cells. The enzyme confined to the 12,000 x g particulate fraction hydrolyses inositol phospholipids, with preference for phosphatidylinositol 4-phosphate (PtdIns(4)P) over phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), exhibiting maximum values of 61 and 15 nmol/min per mg protein, respectively, at a pH optimum of 5.5. The phosphodiesterase, which is strongly Ca2+ dependent with optimal free Ca2+ concentrations between 20 and 100 nM for both substrates, is almost completely inhibited (93-95%) in the presence of 2 mM EGTA. Only the PLC acting on PtdIns(4,5)P2 is significantly activated in the presence of 6-60 microM GTP gamma S. The low extent of enzymatic activity in the presence of 5 mM MgCl2 or chelating agents is suggestive of inositolphosphatase activity which is supported by the determination of small amounts of myo-inositol during HPLC analyses. Both dioleoylglycerol (DAG) and the membrane-permeable 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibit PLC activity, exhibiting IC50 values of 5 microM with PtdIns(4)P and approx. 10 microM with PtdIns(4,5)P2 as substrate and maximum inhibition up to 60% (DAG) and 80% (OAG). These data are indicative of a mechanism of direct negative feedback regulation of the enzyme by diglycerides which may explain the observed long-term effects of OAG on PLC activity in cell culture experiments.
Haeffner
E W
EW
Institut für Zell- und Tumorbiologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Wittmann
U
U
D002118:Calcium / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D004075:Diglycerides / Q000494:pharmacology*; D005246:Feedback; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000502:physiology*; D006801:Humans; D006868:Hydrolysis; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014867:Water / Q000737:chemistry
D004075:Diglycerides; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; C037178:phosphatidylinositol 4-phosphate; D014867:Water; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C038237:1-oleoyl-2-acetylglycerol; D010738:Type C Phospholipases; D002118:Calcium; C013965:diolein