Huennekens F M, Suresh M R, Vitols K S, Henderson G B
Adv Enzyme Regul. 1982;20:389-408. doi: 10.1016/0065-2571(82)90027-9.
L1210 mouse leukemia cells provide a convenient model for examining the mechanisms and components involved in the active transport of various metabolites and drugs. One of these transport systems exhibits a broad specificity for folate compounds, including 4-amino antagonists such as methotrexate. The primary substrate for this system is 5-methyltetra-hydrofolate (Kt = 1 microM), the principal circulating form of the vitamin in mammals. 5-Formyltetrahydrofolate (Kt = 5 microM) and Methotrexate (Kt = 5 microM) are also taken up efficiently, but folate (Kt = 100 microM) is a relatively poor substrate. Vmax for this system is ca. 15 pmoles/min/mg protein. Energy for substrate internalization is provided by an anion-exchange mechanism, and regulation appears to be mediated by cyclic AMP. The system can be inhibited irreversibly by treatment of the cells with photo-activated azido AMP or carbodiimide-activated folate compounds. The latter method allows the membrane-associated binding protein to be labeled in situ, thereby providing a means for identifying it during subsequent solubilization and purification. Guidance for this latter project is provided by previous experience in the purification to homogeneity of a similar folate-binding protein from Lactobacillus casei. L1210 cells also contain an efficient system for the transport of adenine (Kt = 20 microM; Vmax = 200 pmoles/min/mg protein). Uptake of adenine is linked with its conversion to AMP via PRPP-dependent adenine phosphori-bosyltransferase. Pterins, which have a close structural similarity to adenine (as well as to a portion of the folate molecule), are also transported into L1210 cells. Transport of [3H] 6-hydroxymethylpterin (Kt = 20 microM) was inhibited by 6-formylpterin, 6-methylpterin and 6-carboxypterin with Ki values of 42, 100 and 350 microM, respectively. Adenine (Ki = 20 microM) and various other purines were also good inhibitors of pterin transport. Present evidence indicates that adenine and pterins use separate transport systems, but isolation of the components of these systems may further delineate their interrelationships.
L1210小鼠白血病细胞为研究各种代谢物和药物主动转运所涉及的机制及成分提供了一个便利的模型。其中一种转运系统对叶酸化合物具有广泛的特异性，包括甲氨蝶呤等4-氨基拮抗剂。该系统的主要底物是5-甲基四氢叶酸（Kt = 1微摩尔），它是哺乳动物体内维生素的主要循环形式。5-甲酰四氢叶酸（Kt = 5微摩尔）和甲氨蝶呤（Kt = 5微摩尔）也能被高效摄取，但叶酸（Kt = 100微摩尔）是相对较差的底物。该系统的Vmax约为15皮摩尔/分钟/毫克蛋白质。底物内化的能量由阴离子交换机制提供，其调节似乎由环磷酸腺苷介导。用光活化的叠氮腺苷或碳二亚胺活化的叶酸化合物处理细胞可不可逆地抑制该系统。后一种方法可使膜相关结合蛋白在原位被标记，从而为后续溶解和纯化过程中鉴定它提供一种手段。从干酪乳杆菌中纯化出类似叶酸结合蛋白并使其达到同质的先前经验为后一个项目提供了指导。L1210细胞还含有一种高效的腺嘌呤转运系统（Kt = 20微摩尔；Vmax = 200皮摩尔/分钟/毫克蛋白质）。腺嘌呤的摄取与其通过磷酸核糖焦磷酸依赖的腺嘌呤磷酸核糖转移酶转化为AMP有关。与腺嘌呤结构相似（以及与叶酸分子的一部分相似）的蝶呤也能转运到L1210细胞中。[3H] 6-羟甲基蝶呤（Kt = 20微摩尔）的转运分别被6-甲酰蝶呤、6-甲基蝶呤和6-羧基蝶呤抑制，其Ki值分别为42、100和350微摩尔。腺嘌呤（Ki = 20微摩尔）和其他各种嘌呤也是蝶呤转运的良好抑制剂。目前的证据表明腺嘌呤和蝶呤使用不同的转运系统，但分离这些系统的成分可能会进一步阐明它们之间的相互关系。
