Choline metabolism and membrane formation in rat hepatoma cells grown in suspension culture: I. Incorporation of choline into phosphatidylcholine of mitochondria and other membranous structures and effect of metabolic inhibitors

Abstract

The synthesis of phosphatidylcholine and its corporation into membranes of Novikoff rat hepatoma cells (strain N1S1–67) cultivated in suspension culture was investigated using 3H-methyl-choline as specific precursor. When cells were suspended in basal medium containing 0.01 m m choline, the latter was taken up by the cells very rapidly and accumulated intracellularly bound in phosphorylcholine. Results from pulse-chase experiments have shown that phosphorylcholine is a precursor in the synthesis of phosphatidylcholine, probably via the Kennedy pathway, but cytidine diphosphate choline did not accumulate in detectable amounts. The synthesis of phosphorylcholine proceeded 4–5 times more rapidly than its subsequent incorporation into phosphatidylcholine and the cells (1.8 × 10 6 cells per ml) removed over 90% of the choline from the medium within 5–6 hr of incubation at 37 °. The rate of synthesis of phosphatidylcholine by cells was influenced to some extent by the stage of growth of the culture from which the cells were obtained, but was independent of the concentration of choline in the medium between 0.01 and 0.06 m m or the pH between 6.8 and 8.0. Newly synthesized phosphatidylcholine was detectable in cells only as an integral part of cellular membranes. Results from experiments involving differential centrifugation of cell homogenates and isopycnic sucrose density gradient centrifugation of cytoplasmic extracts indicate that about 15% of the total phosphatidylcholine synthesized was associated with the nuclei, 40–50% with the mitochondria, and the remainder with fragments of the plasma membrane and other membranous components with densities (1.13 – 1.15 g/cm 3) lower than that of mitochondria (1.18 g/cm 3). The incorporation of choline into the various membranous structures of the cell followed a similar time course. The density of the mitochondria and other membranous structures was approximately the same whether the cells were incubated in basal medium containing 0.01 or 0.10 m m choline. Inhibition of protein synthesis by treatment of cells with actidione or chloramphenicol did not affect the rates of incorporation of choline into phosphorylcholine or phosphatidylcholine for at least 3 hr. Both processes were slightly inhibited by treating cells with puromycin or actinomycin D.