Characterization of cytosolic phospholipases C from porcine aortic endothelial cells.
Phospholipases C (PLCs) are ubiquitous enzymes which play key roles in the response of cells to extracellular agonists. Endothelial cells are involved in myriad normal and pathophysiologic functions. Although it is known that agonists activate PLCs in endothelial cells, second messengers form, and cellular responses ensue, more knowledge is needed about the specific types of PLCs in these cells. To this end, cytosolic PLCs from porcine aortic endothelial cells were partially purified by ammonium sulfate fractionation and column chromatography on DEAE-Sepharose CL-6B and heparin-agarose. Three PLC isozymes immunologically similar to bovine brain PLC-beta, PLC-gamma, and PLC-delta were identified. The relative levels of PLC activities in the cytosol were: PLC-beta, 50%; PLC-gamma, 44%; PLC-delta, 6%. The level of PLC-beta activity in porcine endothelial cells appeared higher than the levels reported for several established cell lines, suggesting that this enzyme may play a specific role in endothelial cell function. Elution profiles of PLC activity with phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2) as substrate were similar to those with phosphatidylinositol (Ptdlns) as substrate, indicating that cytosolic PLCs hydrolyze both Ptdlns and Ptdlns(4,5)P2 and no Ptdlns(4,5)P2-specific PLC was present in the cytosol. The catalytic properties of the partially purified PLC isozymes from porcine endothelial cells were similar to their counterparts from bovine brain. These include the dependence of hydrolysis of Ptdlns on Ca2+, the optimal Ca2+ concentrations for the hydrolysis of Ptdlns and Ptdlns(4,5)P2, the pH optima, and the stimulatory effects of deoxycholate.
Fu
Y
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Division of Cardiology, New England Deaconess Hospital, Boston, Massachusetts 02215.
Cheng
J X
JX
Hong
S L
SL
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000201:enzymology; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D002417:Cattle; D002478:Cells, Cultured; D002847:Chromatography, Agarose; D003600:Cytosol / Q000201:enzymology*; D003840:Deoxycholic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007527:Isoenzymes / Q000187:drug effects / Q000302:isolation & purification*; D013552:Swine; D010738:Type C Phospholipases / Q000187:drug effects / Q000302:isolation & purification*
D007527:Isoenzymes; D003840:Deoxycholic Acid; D010738:Type C Phospholipases; D002118:Calcium