Overexpression of rat liver CTP:phosphocholine cytidylyltransferase accelerates phosphatidylcholine synthesis and degradation.
Two rat liver cDNAs encoding CTP:phosphocholine cytidylyltransferase (CT-1 and CT-2) were expressed in COS cells. The specific activity of CT in the microsomes increased approximately 20- or 100-fold after transfection with CT-1 or CT-2, respectively, but there was only a 3-5 fold increase in the rate of [3H]choline or [3H]glycerol incorporation into phosphatidylcholine (PC). The phosphocholine pool decreased approximately 40% in keeping with a stimulation of the CT-catalyzed reaction. The CDP-choline pool increased 12-fold suggesting that the conversion of CDP-choline to PC, catalyzed by cholinesphosphotransferase, could not keep pace with the CT-catalyzed reaction. This could account for the discrepancy between the increases in the amount of active (membrane-bound) CT and the rate of PC synthesis. Incubation of CT-transfected cells with sodium oleate to increase the supply of cellular diacylglycerol resulted in a further 2-fold increase in the rate of PC synthesis. This suggests that the diacylglycerol supply may be a limiting factor in the degree of stimulation of PC synthesis in CT-transfected COS cells. Despite the increased rate of PC synthesis, the total cellular PC mass increased only 17%, due to a 3-fold acceleration of the PC degradation rate. To determine which degradative pathway for PC was accelerated in the CT-transfected cells, we measured the pool sizes of several catabolites. Neither diacylglycerol nor phosphatidic acid mass was altered. The pool of glycerophosphocholine (GPC) was increased approximately 4-fold, and there was elevated release of GPC from the CT-transfected cells. The turnover of choline in GPC and lyso-PC was very slow compared with that of choline, phosphocholine, or CDP-choline, suggesting that GPC and lyso-PC were derived from slowly degraded choline-labeled PC. The metabolism of GPC and lyso-PC was stimulated in the cells over-expressing CT. These data suggest that PC synthesis and degradation are coordinated and that PC catabolism involving PC-->lyso-PC-->GPC is accelerated in COS cells overexpressing CT.
Walkey
C J
CJ
Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Kalmar
G B
GB
Cornell
R B
RB
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002794:Choline / Q000378:metabolism; D019916:Choline-Phosphate Cytidylyltransferase; D003600:Cytosol / Q000201:enzymology; D005990:Glycerol / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism*; D051381:Rats; D014162:Transfection
D009829:Oleic Acids; D010713:Phosphatidylcholines; D019301:Oleic Acid; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D002794:Choline; D005990:Glycerol