A rapid method for the determination of CTP and phosphocholine in rat liver and baby hamster kidney 21 cells.
A rapid and sensitive assay for CTP and phosphocholine was required for us to determine the concentration of these compounds in tissues and cell cultures. Such a procedure was devised with CTP:phosphocholine cytidylyltransferase, an enzyme which is highly specific for CTP and phosphocholine. The 0--22% ammonium sulfate precipitate of a cytosolic extract from rat liver was used as the source of the enzyme. The amount of CTP in an extract was estimated by the conversion of [3H]phosphocholine to 3H-labelled CDP-choline. Similarly, the concentration of phosphocholine was estimated by the formation of 3H-labelled CDP-choline from 3H-labelled CTP. The conversion of CTP and phosphocholine to CDP-choline was 90% when inorganic pyrophosphatase was added to the incubations. The formation of CDP-choline was linear between 1 and 10 nmol of CTP or phosphocholine. The concentration of CTP was determined in rat liver (62 nmol/g wet weight) and baby hamster kidney 21 (BHK-21) cells (161 nmol/g wet weight). The concentration of phosphocholine in rat liver was 1.16 mumol/g wet weight whereas in BHK-21 cells it was much less (69 nmol/g wet weight). By this procedure, it may be possible to establish the importance of CTP and phosphocholine in the control of phosphatidylcholine biosynthesis.
D002460:Cell Line; D002794:Choline / Q000031:analogs & derivatives*; D003570:Cytidine Triphosphate / Q000032:analysis* / Q000378:metabolism; D003597:Cytosine Nucleotides / Q000032:analysis*; D008099:Liver / Q000032:analysis*; D008722:Methods; D009713:Nucleotidyltransferases / Q000378:metabolism; D010767:Phosphorylcholine / Q000032:analysis* / Q000378:metabolism
D003597:Cytosine Nucleotides; D010767:Phosphorylcholine; D003570:Cytidine Triphosphate; D009713:Nucleotidyltransferases; D002794:Choline