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Role of Endocytosis and Lysosomal pH in Uptake of N-(Phosphonacetyl)-L-aspartate and Its Inhibition of Pyrimidine Synthesis¹

Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103

The mechanism of uptake and retention of N-(phosphonacetyl)-L-aspartate (PALA) was examined. Uptake of [³H]PALA by Ehrlich ascites tumor cells appeared to be biphasic. A small, variable quantity of PALA associated with cells within 5 min; the significance of this rapid uptake component is unclear. Between 15 min and 5 h, uptake was linear and consistent from experiment to experiment. The properties of the slow phase of PALA uptake are consistent with fluid-phase endocytosis. The intracellular PALA concentration approached the extracellular level very slowly, at a rate of approximately 1%/hr. The velocity of PALA uptake in these cells was proportional to the concentration in the media from 10^-6 to 10^-2 M. Uptake of PALA was identical to that of the extracellular marker inulin. Uptake of both PALA and inulin was inhibited by colchicine and stimulated by phorbol myristate acetate. The microtubule antagonist and the phorbol ester are known to, respectively, inhibit and stimulate endocytosis in other cell types. Phorbol myristate acetate enhanced the ability of PALA to inhibit incorporation of [¹⁴C]bicarbonate into pyrimidine nucleotides, presumably through an increase in PALA uptake. This inhibitory action of PALA was almost completely blocked by two agents known to neutralize lysosomal pH, NH₄Cl and methylamine. These results suggest that intracellular PALA is initially compartmentalized in a pinosomal vesicle which may later fuse with cellular lysosomes. Neutralization of lysosomal pH prevents the protonation of some or all of the four negatively charged groups found in the structure of PALA which may be necessary for its diffusion across the lysosomal membrane and eventual inhibition of aspartate transcarbamylase in the cytoplasm. Since partitioning of the fully charged molecule into the lipid phase of the plasma membrane for diffusion out of the cell should be minimal, the effects of PALA on cellular metabolism are expected to be prolonged.

¹ This research was supported by Grant CH-206 from the American Cancer Society, a Pharmaceutical Manufacturer's Association Foundation Research Starter grant, and by a Venture Grant of the Bowman Gray School of Medicine.

² To whom requests for reprints should be addressed.

Received 9/ 9/83. Accepted 10/25/83.