Henderson G B, Zevely E M, Huennekens F M
J Biol Chem. 1979 Oct 25;254(20):9973-5.
Methotrexate transport in L1210 cells is mediated by a carrier protein that can bind organic and inorganic phosphate compounds in addition to the various folate substrates. The photoaffinity labeling agent, 8-azidoadenosine 5'-monophosphate (8-azido-AMP), also interactis (Ki = 140 microM) with the receptor site for this transport system, and upon irradiation with ultraviolet light, irreversibly inhibits methotrexate uptake. Protection against this inactivation is afforded by either a substrate (methotrexate) or a competitive inhibitor (inorganic phosphate). The light-induced reaction proceeds rapidly (t1/2 = 2 min at 23 degrees C under the conditions described) and produces half-maximal reduction in the transport rate when the 8-azido-AMP concentration is 65 microM. complete photoinactivation of methotrexate transport could not be obtained from a single exposure to 8-azido-AMP (up to 1.0 mM), but it could be achieved by the repetitive illumination of cells in a fresh medium. The phosphate and folate/adenine transport systems of L1210 cells are not affected by irradiation in the presence of 8-azido-AMP.
甲氨蝶呤在L1210细胞中的转运由一种载体蛋白介导，该载体蛋白除了能结合各种叶酸底物外，还能结合有机和无机磷酸盐化合物。光亲和标记剂8-叠氮腺苷5'-单磷酸（8-azido-AMP）也与该转运系统的受体位点相互作用（Ki = 140 microM），并且在紫外线照射下，不可逆地抑制甲氨蝶呤的摄取。底物（甲氨蝶呤）或竞争性抑制剂（无机磷酸盐）可提供针对这种失活的保护作用。光诱导反应进行迅速（在所描述的条件下，23℃时t1/2 = 2分钟），当8-azido-AMP浓度为65 microM时，转运速率降低一半。单次暴露于8-azido-AMP（高达1.0 mM）无法使甲氨蝶呤转运完全光失活，但通过在新鲜培养基中对细胞进行重复照射可以实现。L1210细胞的磷酸盐和叶酸/腺嘌呤转运系统在8-azido-AMP存在下不受照射影响。
