Henderson G B, Montague-Wilkie B
Biochim Biophys Acta. 1983 Oct 26;735(1):123-30. doi: 10.1016/0005-2736(83)90267-5.
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 degrees C, inhibition was rapid (t1/2 less than 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 degrees C. Inactivation in this case was qualitatively similar to the corresponding process at 37 degrees 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 degrees 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.
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生成甲氨蝶呤的活化酯，它是L1210细胞中甲氨蝶呤转运的一种强效不可逆抑制剂。在用该试剂在37℃处理的细胞中，抑制作用迅速（半衰期小于1分钟），在pH 6.8时最佳，抑制剂浓度为20 nM时达到半数最大抑制，且在高浓度试剂时完全抑制。预处理步骤中加入过量甲氨蝶呤可保护转运系统不被灭活，这表明了其特异性。在4℃暴露于该试剂的细胞中也观察到不可逆抑制。在这种情况下的失活在性质上与37℃时的相应过程相似；它迅速出现，在20 nM时达到半数最大抑制，并且可以通过加入高浓度底物来防止。然而，即使在含有过量或多次添加试剂的样品中，抑制程度最高也仅达到75%。后一发现表明，在4℃时转运蛋白以两种形式存在，一种（占总数的75%）含有可被活化酯作用的结合位点，另一种（占总数的25%）含有不可作用的位点。这些位点被认为分别是具有外向和内向取向的转运蛋白。
