PERMEATION AS RATE-LIMITING STEP IN PHOSPHORYLATION OF URIDINE AND CHOLINE AND THEIR INCORPORATION INTO MACROMOLECULES BY NOVIKOFF HEPATOMA CELLS . COMPETITIVE INHIBITION BY PHENETHYL ALCOHOL, PERSANTIN, AND ADENOSINE

The incorporation of uridine or choline into intracellular free phosphorylated intermediates by exponentially growing Novikoff rat hepatoma cells in suspension culture follows simple Michaelis-Menten kinetics. The apparent Vm’s and Km’s for the incorporation of uridine or choline by whole cells, however, are at least one order of magnitude lower than those for the phosphorylation of the substrates by cell-free extracts. Further, the incorporation of uridine by whole cells is inhibited competitively by adenosine, persantin, and phenethyl alcohol, whereas the phosphorylation of uridine by cell-free extracts is inhibited noncompetitively by phenethyl alcohol and is unaffected by adenosine or persantin. Phenethyl alcohol is also a competitive inhibitor for choline incorporation by whole cells and a noncompetitive inhibitor for choline kinase as measured in cell-free extracts. Both the uridine and choline kinase activities of the cells are located in the soluble portion of the cytoplasm and isolated fragments of plasma membrane are free of kinase activities. The overall results are interpreted to indicate that the transport of uridine and choline into the cell are reactions distinct from phosphorylation and that permeation is the rate-limiting step in the incorporation of these precursors by whole cells into the intracellular pools of phosphorylated intermediates. The inhibition of the transport of uridine or choline into the cells results in a proportional inhibition of the incorporation of the precursors into ribonucleic acid and membrane phosphatidylcholine, respectively. Thus the rate of incorporation of these precursors into macromolecules is also limited by the rate with which they enter the cells. © 1969, American Chemical Society. All rights reserved.