Regulation of rat liver cytosolic CTP: phosphocholine cytidylyltransferase by phosphorylation and dephosphorylation.

Rat liver cytosolic CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) activity was elevated by incubation of cytosol (1.7-fold at 37 "C for 10 min or 5.5fold at 20 "C for 5 h).At 37 "C, this activation was prevented in a time-and concentration-dependent manner when 14 m M magnesium and 0.5 m~ ATP were added to the cytosol.Titration of cytosol with protein kinase inhibitors from rabbit muscle and beef and porcine heart for 10 min at 37 "C permitted a 5-fold activation in the presence of Mg-ATP, while control cytosol was stimulated 2.2-fold.At 20 "C, the activation of cytidylyltransferase was reduced &fold by the presence of 20 m~ NaF.The K,,, of the Mg-ATP-treated cytidylyltransferase for phosphocholine was unchanged compared to controls, although substrate inhibition was apparent for phosphocholine concentrations exceeding 1 m ~.The K,,, of the Mg.ATP-treated cytidylyltransferase for CTP was as high as 5 mM, while protein kinase inhibitor-treated cytidylyltransferase had a K , of approximately 0.35 m ~.The Mg-ATP-treated cytidylyltransferase activity could be elevated 6-fold by phospholipid prepared from total rat liver and the K,,, for CTP reduced to 0.2 mM.In contrast, cytidylyltransferase activity stimulated by protein kinase inhibitor was reduced 25% by phospholipid.Collectively, these data implicate cytidylyltransferase as an enzyme susceptible to regulation by a phosphorylation-dephosphorylation cycle.Phosphatidylcholine is synthesized primarily via the CDPcholine route in rat liver and other systems (1).Numerous studies have identified CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) as a key regulatory enzyme for this pathway (2-13).This enzyme is ambiquitous ( 14) in that it is recovered in both the cytosolic and microsomal fractions of the liver homogenate (15).In the microsomes, the cytidylyltransferase is located on the cytoplasmic side of the sealed fragments of endoplasmic reticulum ( 16).The distribution of enzyme activity between these fractions is dependent on the homogenization medium and method of centrifugation (17).When liver is homogenized in isotonic 0.9% NaCl solution, the majority of the enzyme activity resides in the cytosolic fraction (18).The cytidylyltransferase in fresh rat liver cytosol has a M , of 2 X lo5 and has been designated the L-form' (15).When cytosol