Identification of phosphatidylcholine-selective and phosphatidylinositol-selective phospholipases D in Madin-Darby canine kidney cells.
Intact cells and cell-free systems were employed to characterize phospholipase D (PLD) activity in Madin-Darby canine kidney (MDCK) cells. In cells prelabeled with [3H]glycerol, 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited phosphatidylcholine (PC) hydrolysis by PLD, as shown by the prolonged formation of [3H]phosphatidylethanol (PEt) and an accompanying decrease in [3H]PC. In contrast, bradykinin elicited rapid formation of [3H]PEt (approximately 1 min) accompanied by a decrease in [3H]phosphatidylinositol (PI). When the agonists were administered simultaneously, [3H]PEt formation was biphasic. In cells prelabeled with [3H] choline, at times less than 1 min, bradykinin failed to induce significant change in [3H]choline release. Bradykinin-induced formation of [3H]PEt in the [3H]glycerol-labeled cells was strictly dependent on extracellular Ca2+, whereas TPA-induced formation of [3H]PEt did not require extracellular Ca2+. Cell-free assays for PLD were used to assess the enzyme location, substrate specificity, and cofactor requirements. The PC-PLD activity (PEt formation) against [3H]stearoyl-PC was primarily localized in the 440 x g pellet (membrane- and nuclear-associated), preferred PC as a substrate, required detergent, and was not influenced by Ca2+ at low concentrations but was inhibited by Ca2+ in excess of 0.5 mM. The PI-PLD activity against [3H]stearoyl-PI was found largely in the 100,000 x g supernatant (cytosol), was strictly Ca(2+)-dependent, and did not require detergent. From these data, we conclude that MDCK cells contain two PLD subtypes: 1) a membrane-associated, PC-selective enzyme that responds to TPA resulting in prolonged hydrolysis of PC (the PC-PLD is Ca(2+)-independent, but requires detergent); 2) a cytosolic, PI-selective enzyme that responds rapidly but transiently to bradykinin (the PI-PLD requires Ca2+ but not detergent).
Huang
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Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157-1016.
Wykle
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Daniel
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LW
Cabot
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MC
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002458:Cell Fractionation; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007668:Kidney; D007700:Kinetics; D010713:Phosphatidylcholines / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010739:Phospholipase D / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D003902:Detergents; D007527:Isoenzymes; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D002118:Calcium