A direct method for the simultaneous measurement of ceramide and phospholipase D activity.
Both ceramide and phospholipase D (PLD) have important roles in a variety of signal transduction pathways. Recent evidence suggests that ceramide is a novel second messenger with specific biological effects. Publications in this field have increased rapidly in the last few years. However, a method to directly and rapidly measure cermide production has been lacking. Herein, we report on a novel, inexpensive, direct and rapid assay for the measurement of ceramide and the simultaneous measurement of PLD activity. This method uses labeling of cells with [(14)C]myristic acid and a TLC solvent of ethyl acetate/acetic acid/trimethylpentane. This method avoids the loss of radioactivity and variability due to changes in DAG kinase activity that are associated with the commonly-used DAG kinase assay.
Liu
G
G
Department of Cellular and Molecular Medicine, Kidney Research Center, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
Kleine
L
L
Hébert
R L
RL
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D002518:Ceramides / Q000032:analysis* / Q000378:metabolism; D002855:Chromatography, Thin Layer; D019852:Diacylglycerol Kinase / Q000378:metabolism; D019814:Myristic Acid / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010739:Phospholipase D / Q000032:analysis* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011817:Rabbits; D012997:Solvents; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013211:Staphylococcus aureus
D002250:Carbon Radioisotopes; D002518:Ceramides; D010714:Phosphatidylethanolamines; D010761:Phosphorus Radioisotopes; D012997:Solvents; D019814:Myristic Acid; D019852:Diacylglycerol Kinase; D013108:Sphingomyelin Phosphodiesterase; D010739:Phospholipase D; D001920:Bradykinin