Journal of Lipid Research, Vol. 12, 715-724, November 1971
Copyright © 1971 by Lipid Research, Inc.

Choline metabolism and membrane formation in rat hepatoma cells grown in suspension culture. III. Choline transport and uptake by simple diffusion and lack of direct exchange with phosphatidylcholine

Peter G. W. Plagemann

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

The initial rate of incorporation of methyl-labeled choline into the acid-soluble pool (phosphorylcholine) of Novikoff hepatoma cells growing in suspension culture was investigated as a function of the choline concentration in the medium. Below, but not above, 20 µm, choline incorporation followed simple Michaelis-Menten kinetics at 24, 33, or 37°C with an apparent K_m of 4-7 µm, and the V_max values decreased with a Q₁₀ of about 2.3 with a decrease in temperature. Between 20 and 500 µm, on the other hand, the rate of incorporation increased linearly with an increase in choline concentration in the medium, and the increase in incorporation rate with increase in choline concentration was about the same at all temperatures tested. The data suggest that at low concentrations choline is taken up mainly by a transport reaction, whereas at concentrations above 20 µm, simple diffusion becomes the principal mode of uptake. The energy of activation for choline transport was estimated from an Arrhenius plot of the V_max values as 67,000 J (16 kcal)/mole.

At concentrations below 20 µm, choline incorporation into membrane phosphatidylcholine also followed simple Michaelis-Menten kinetics, and the apparent K_m was about the same as that for choline transport. The data support the conclusion that the transport of choline into the cell is the rate-limiting step in the conversion of choline to phosphorylcholine and its incorporation into phosphatidylcholine. At concentrations above 100 µm, on the other hand, the ultimate rate of choline incorporation into phosphatidylcholine was independent of the choline concentration in the medium or the intracellular level of phosphorylcholine. Further, the rate of turnover of the choline moiety of phosphatidylcholine (half-life, 20-24 hr) either in whole cells or during incubation of isolated membrane fractions was unaffected by the presence of an excess of choline in the medium. The overall results indicate that a direct exchange between free choline and the choline moiety of phosphatidylcholine does not play a significant role in the incorporation of choline into phosphatidylcholine by Novikoff cells or in the turnover of the choline moiety of phosphatidylcholine, and that labeled choline therefore is a useful precursor in studying the synthesis and turnover of membrane phosphatidylcholine in these cells.

Supplementary key words choline uptake • phosphatidylcholine synthesis and turnover • Novikoff rat hepatoma cells • Michaelis-Menten kinetics

Submitted on March 10, 1971
Accepted on June 24, 1971

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