Carrier-mediated transport of the folic acid analogue, methotrexate, in the L1210 leukemia cell.
Carrier-mediated transport of the folic acid analogue, methotrexate, in the L1210 leukemia cell.
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DOI：
10.1016/s0021-9258(18)91984-8
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发表时间：
1968-10
期刊：
影响因子：
--
通讯作者：
Norman;S. Lichtenstein;Vincent;-T.;Oliverio
中科院分区：
文献类型：
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作者：
Norman;S. Lichtenstein;Vincent;-T.;Oliverio
本文研究了叶酸结构类似物甲氨蝶呤（4-氨基-10-甲基蝶酰谷氨酸）在L1210小鼠白血病细胞中的转运。(a)有一个基本上是不可交换的细胞内部分可能绑定的酶二氢叶酸还原酶。(b)剩余的细胞内甲氨蝶呤可快速交换，可能以抗肿瘤活性状态存在于细胞水中。此外，有一个小的快速交换组分的甲氨蝶呤与细胞，具有松散的吸附在细胞膜上或附近的特点。甲氨蝶呤在该细胞系统中的代谢可以忽略不计，因为细胞内存在与甲氨蝶呤紧密结合的位点，在这些位点饱和之前，游离的细胞内甲氨蝶呤不会出现，因此外排可以忽略不计。这允许单向上升速度的精确测量。单向流入过程的主要部分表现出Michaelis-Menten动力学与高度的结构特异性，竞争性抑制叶酸和亚叶酸，和显着的温度依赖性。这些竞争性物质的过量添加到细胞在稳定状态下与细胞外甲氨蝶呤导致细胞内甲氨蝶呤的countertransport.Electrochemical电位梯度游离细胞内甲氨蝶呤被证明在低水平的细胞外甲氨蝶呤和细胞内甲氨蝶呤浓度在平衡时表现出Michaelis-Menten动力学。米氏常数和最大速度的单向流出过程进行了估计，当涉及到这些参数的单向流入，是兼容的积累system.These研究表明，甲氨蝶呤运输在L1210细胞是载体介导的和上坡。甲氨蝶呤具有几个品质，这使得它成为一个有价值的“模型基板”的运输特性的研究叶酸及其天然存在的derivatives.The暗示这些发现相对于更有效的叶酸拮抗剂的发展被认为是。
Transport of methotrexate (4-amino-10-methylpteroyl-glutamic acid), a structural analogue of folic acid, was studied in L1210 mouse leukemia cells grown in tissue culture.Intracellular methotrexate was present in two states. (a) There was an essentially nonexchangeable intracellular fraction probably bound to the enzyme dihydrofolate reductase. (b) The remaining intracellular methotrexate was rapidly exchangeable and likely present in the cell water in an osmotically active state. In addition, there was a small rapidly exchanging component of methotrexate associated with the cells which had the characteristics of a loose adsorption on or near to the cell membrane. Methotrexate was shown to be negligibly metabolized by this cell system.Because of the presence of tight intracellular binding sites for methotrexate, prior to saturation of these sites, free intracellular methotrexate does not appear and consequently efflux is negligible. This permits the accurate measurement of unidirectional uptake velocities. The major portion of the unidirectional influx process demonstrated Michaelis-Menten kinetics with a high degree of structural specificity, competitive inhibition by folic and folinic acids, and marked temperature dependence. Addition of these competing substances in excess to cells at the steady state with extracellular methotrexate leads to countertransport of intracellular methotrexate.Electrochemical potential gradients for free intracellular methotrexate were demonstrated at low levels of extracellular methotrexate and intracellular methotrexate concentrations at equilibrium exhibited Michaelis-Menten kinetics.Efflux of intracellular methotrexate was not first order and appeared to be partially saturated. The Michaelis constant and maximum velocity for the unidirectional efflux process were estimated, and when related to these parameters for the unidirectional influx, were compatible with an accumulating system.These studies indicate that methotrexate transport in the L1210 cell is carrier-mediated and uphill. Methotrexate exhibits several qualities which make it a valuable “model substrate” for the study of the transport characteristics of folic acid and its naturally occurring derivatives.The implication of these findings with respect to the development of more effective folic acid antagonists is considered.