Studies on choline permeation through the plasma membrane and its incorporation into phosphatidyl choline of Ehrlich-Lettre-ascites tumor cells in vitro

The initial rate of incorporation of 14C or 3H‐labeled choline into Ehrlich‐Lettre ascites cells of the glycogen‐free strain seven days after inoculation was investigated in vitro. At choline concentrations in the medium between 6 to 30 μM and 100 to 500 μM the choline uptake by the cells followed Michaelis‐Menton kinetics with V values between 31 to 100 and 59 to 500 pmol per minute at a given cell density, and average Q10‐values of 2.1 at the high and of 2.4 at the low choline molarity. The Km‐values increased from 27 μM to 58.8 μM at low and from 0.11 mM to 0.22 mM at high choline concentrations over a temperature range between 15°C and 37°C. Arrhenius plot of the V values gave two lines, one with a transition temperature at 25°C at low and one straight line at high choline concentrations, from which the energy of activation for choline uptake was determined to be 16 kcal/mol. It is assumed that two systems exist for the choline uptake by the ascites cells. One, operative at low substrate concentrations, which is saturable and probably is to be classified as a carrier‐mediated facilitated diffusion process, can be strongly inhibited by deoxyglucose or 2,4‐dinitrophenol and also by substrate analogues such as chlorocholine or benzoylcholine. Ouabain affects this system to a lesser extent. The other system functioning at high choline concentrations may be a simple diffusion process, which is little inhibited by substrate analogues, ouabain and deoxyglucose; however, it is also inhibited by 2,4‐dinitrophenol and p‐chloromercuribenzoate. Choline incorporation into the acid‐insoluble material (lecithin) gave linear Michaelis‐Menton kinetics at the low and the high substrate concentration respectively. Km‐values decreased with an increase in temperature at low and increased with rising temperature at high substrate concentrations thus reflecting a close relationship between choline uptake and its metabolism. Labeling of lecithin choline in the various subcellular fractions under the conditions of the functioning of a carrier‐mediated process was in the order: mitochondria (50%) > plasma membranes (25%) > nuclei (14%) > microsomes (9%) > supernatant (1.5%). Treatment of the cells with p‐chloromercuribenzoate or heat shock at 50°C markedly reduced the choline uptake and concomitantly its conversion into lecithin. Kinetic analysis revealed that the inhibitory effect of p‐chloromercuribenzoate was competitive and that of the heat shock non‐competitive in nature. Further the choline uptake by the cells was found to be the rate‐limiting step, since the rate of choline phosphorylation was determined by the extracellular choline concentration. Pulse chase experiments showed a rapid turnover of the choline moiety with a concomitant increase in activity of the lecithin fraction and little change within the choline phosphate pool.