Irreversible inhibitors of methotrexate transport in L1210 cells. Characteristics of inhibition by an N-hydroxysuccinimide ester of methotrexate.
Irreversible inhibitors of methotrexate transport in L1210 cells. Characteristics of inhibition by an N-hydroxysuccinimide ester of methotrexate.
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L1210 细胞中甲氨蝶呤转运的不可逆抑制剂。
DOI：
10.1016/0005-2736(83)90267-5
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Montague-Wilkie,B
中科院分区：
文献类型：
--
作者：
Henderson,GB;Montague-Wilkie,B
摘要 甲氨蝶呤、1-乙基-3-(3-二甲基氨基丙基)碳二亚胺和 N-羟基琥珀酰亚胺反应形成甲氨蝶呤的活化酯，它是 L1210 细胞中甲氨蝶呤转运的有效不可逆抑制剂。在 37° C 下用该试剂处理的细胞中，抑制速度很快（t 1 2 < 1 分钟），在 pH 6.8 时达到最佳抑制效果，在抑制剂浓度为 20 nM 时达到半最大抑制，并在高水平的试剂下完全抑制。预处理步骤中添加的过量甲氨蝶呤可防止转运系统失活，这一事实表明了特异性。在 4°C 下暴露于该试剂的细胞中也观察到不可逆的抑制。这种情况下的灭活在质量上与 37°C 下的相应过程相似；它出现得很快，在 20 nM 时为最大浓度的一半，并且可以通过添加高浓度的底物来阻止。然而，即使在含有过量或多次添加试剂的样品中，抑制程度最多也仅达到 75%。后一发现表明，在 4°C 时，转运蛋白以两种形式存在，一种（占总数的 75%）含有活性酯可接近的结合位点，另一种（占总数的 25%）含有不可接近的位点。这些位点的身份被认为是分别具有向外和向内方向的转运蛋白。
Abstract Methotrexate, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide react to form an activated ester of methotrexate which is a potent irreversible inhibitor of methotrexate transport in L1210 cells. In cells treated with the reagent at 37° C, inhibition was rapid (t 1 2< 1 min), optimal at pH 6.8, half-maximal at an inhibitor concentration of 20 nM, and complete at high levels of the reagent. Specificity was indicated by the fact that excess methotrexate added during the pretreatment step protected the transport system against inactivation. Irreversible inhibition was also observed in cells exposed to the reagent at 4° C. Inactivation in this case was qualitatively similar to the corresponding process at 37° C; it appeared rapidly, was half-maximal at 20 nM, and could be prevented by the addition of high concentrations of the substrate. The extent of the inhibition, however, reached a maximum of only 75%, even in samples containing excess or multiple additions of reagent. The latter findings suggest that at 4° C the transport protein exists in two forms, one (75% of the total) containing binding sites which are accessible to the active ester, and the other (25% of the total) with inaccessible sites. The identity of these sites is suggested to be transport proteins which have outward and inward orientations, respectively.