White J C, Hines L H
Cancer Res. 1984 Feb;44(2):507-13.
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.
研究了N-（膦酰乙酰基）-L-天冬氨酸（PALA）的摄取和潴留机制。艾氏腹水瘤细胞对[3H]PALA的摄取似乎呈双相性。在5分钟内，少量可变的PALA与细胞结合；这种快速摄取成分的意义尚不清楚。在15分钟至5小时之间，摄取呈线性，且不同实验之间一致。PALA摄取慢相的特性与液相内吞作用一致。细胞内PALA浓度非常缓慢地接近细胞外水平，速率约为1%/小时。这些细胞中PALA的摄取速度与培养基中10（-6）至10（-2）M的浓度成正比。PALA的摄取与细胞外标记物菊粉的摄取相同。秋水仙碱抑制PALA和菊粉的摄取，佛波酯肉豆蔻酸酯刺激其摄取。已知微管拮抗剂和佛波酯分别抑制和刺激其他细胞类型的内吞作用。佛波酯肉豆蔻酸酯增强了PALA抑制[14C]碳酸氢盐掺入嘧啶核苷酸的能力，可能是通过增加PALA的摄取。PALA的这种抑制作用几乎完全被两种已知可中和溶酶体pH值的试剂氯化铵和甲胺阻断。这些结果表明，细胞内PALA最初被分隔在吞噬泡中，吞噬泡可能随后与细胞溶酶体融合。溶酶体pH值的中和可防止PALA结构中四个带负电荷基团中的部分或全部质子化，这可能是其跨溶酶体膜扩散并最终抑制细胞质中天冬氨酸转氨甲酰酶所必需的。由于完全带电的分子进入质膜脂质相以扩散出细胞的分配应最小，因此预计PALA对细胞代谢的影响会延长。
