Ceramide quantitation: evaluation of a mixed micellar assay using E. coli diacylglycerol kinase.
The phosphorylation of ceramide solubilized in octylglucoside/phosphatidylglycerol mixed micelles by E. coli diacylglycerol kinase was evaluated. Ceramide containing non-hydroxy fatty acids appeared to be phosphorylated quantitatively over a broad range from 25 to 2000 pmoles. If a 2-hydroxy fatty acid was present in the ceramide molecule, phosphorylation was not quantitative. When applied to cellular lipid extracts, TLC of the phosphorylated products is needed to separate ceramide-phosphates from the other labelled compounds (i.e. phosphatidate and lysophosphatidate) and revealed the presence of ceramides containing long and very long chain fatty acids. The mass levels of these ceramides in different cultured cells varied between 0.2 to 0.6 mol% (normalized to phospholipids). Changes in these levels were observed under different stress conditions such as heat treatment or addition of DMSO or detergents to the cell cultures.
Van Veldhoven
P P
PP
Katholieke Universiteit Leuven, Campus Gasthuisberg, Afdeling Farmakologie, Belgium.
Bishop
W R
WR
Yurivich
D A
DA
Bell
R M
RM
D000818:Animals; D001383:Azetidinecarboxylic Acid / Q000494:pharmacology; D002460:Cell Line; D002518:Ceramides / Q000032:analysis* / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003840:Deoxycholic Acid / Q000494:pharmacology; D003902:Detergents / Q000378:metabolism; D019852:Diacylglycerol Kinase; D004926:Escherichia coli / Q000201:enzymology*; D005960:Glucosides / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008823:Micelles; D010715:Phosphatidylglycerols / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism
D002518:Ceramides; D003902:Detergents; D005960:Glucosides; D008823:Micelles; D010715:Phosphatidylglycerols; D003840:Deoxycholic Acid; C018619:octyl-beta-D-glucoside; D001383:Azetidinecarboxylic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase; D013110:Sphingosine