Cytidylyltransferase translocation onto endoplasmic reticulum and increased de novo synthesis without phosphatidylcholine accumulation in Krebs-II ascite cells

Abstract

Addition of oleic acid to Krebs-II cells stimulated by 9-fold [ 3H]choline incorporation into choline glycerophospholipids without affecting the selective incorporation of the precursor into diacyl subclass (90% of total [ 3H]choline glycerophospholipids). The total activity of cytidylyltransferase (E.C. 2.7.7.15), the regulatory enzyme of choline glycerophospholipid synthesis, was increased in the particulate fraction at the expense of cytosol. Free [ 3H]oleic acid was also associated with the particulate fraction. Subcellular fractionation of membranes on Percoll gradient, indicated that the endoplasmic reticulum, which contained 90% of total cell free oleic acid, was the unique target for the translocation of cytidylyltransferase. [ 3H]oleic acid was incorporated almost exclusively into phosphatidylcholine and corresponded to a synthesis of 9 nmol/h per 10 6 cells. Based on [ 3H]choline incorporation a net synthesis of 22 nmol/h per 10 6 cells was determined. However, oleic acid treatment did not change the total amount of phosphatidylcholine (45 nmol/10 6 cells) and other phospholipids; also no modification in the subcellular distribution of phospholipids was observed. It is concluded that the stimulation of the de novo synthesis of phosphatidylcholine which involves translocation of cytidylyltransferase onto the endoplasmic reticulum, is accompanied by a renewal of their polar head group. Also exogenous oleic acid induces an enhanced fatty acid turnover, highly specific for phosphatidylcholine. Therefore, Krebs-II cells exhibited a high degree of regulation of their phosphatidylcholine content, suggesting a parallel stimulation of both synthesis and degradation.