Phospholipase D hydrolysis of choline phosphoglycerides is selective for the alkyl-linked subclass of Madin-Darby canine kidney cells.
Madin-Darby canine kidney (MDCK) cells were used to study the synthesis of diglycerides from choline phospholipids (PC) in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). In this system, diglyceride formation was blocked in the presence of ethanol (0.5%), and a corresponding amount of phosphatidylethanol (PEt) was formed, indicating that phospholipase D is responsible for the diglyceride production. Analysis of the subclasses of phosphatidylethanol revealed 1-O-alkyl-(alkyl), 1-O-alk-1'-enyl-(alkenyl), and 1-acyl species of PEt (38.0, 8.3, and 53.7%, respectively). The molecular species of the alkyl-PEt most closely matched the alkyl-PC. No change in the relative amounts of alkyl- versus acyl-PEt was observed with time after stimulation. Comparison of the alkyl content of PEt (38.0%) and the parent PC (15.2%) indicated a marked selectivity for the alkyl subclass of PC. A cell-free assay (Huang, C., Wykle, R. L., Daniel, L. W., and Cabot, M. C. (1992) J. Biol. Chem. 267, 16859-16865) for phospholipase D was also used to confirm the selectivity of the enzyme for alkyl-PC versus acyl-PC. The predominant molecular species of PEt contained saturated acyl or alkyl chains in position-1 and monounsaturated residues in position-2 accounting for approximately 50% of the total PEt. 1-O-Octadecyl-2-oleoyl-sn-glycerol, a representative alkyl molecular species, was synthesized and tested for its effect upon protein kinase C derived from MDCK cells. This alkyl-diglyceride (DG) neither stimulated protein kinase C nor inhibited its activation by diacylglycerol. In summary, TPA-stimulated phospholipase D is selective for the alkyl-PC subclass in MDCK cells. The alkyl-DG subsequently formed does not appear to function as a second-messenger in activating protein kinase C.
Daniel
L W
LW
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157.
Huang
C
C
Strum
J C
JC
Smitherman
P K
PK
Greene
D
D
Wykle
R L
RL
D000478:Alkylation; D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D020404:Glycerophospholipids; D006868:Hydrolysis; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D010712:Phosphatidic Acids / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; C051521:phosphatidylethanol; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate