Matherly L H, Czajkowski C A, Angeles S M
Developmental Therapeutics Program, Michigan Cancer Foundation, Detroit 48201.
Cancer Res. 1991 Jul 1;51(13):3420-6.
A K562 human erythroleukemia line (designated K562.4CF) was selected for increased tetrahydrofolate cofactor transport in a growth-limiting concentration (0.4 nM) of (6R,S)-5-formyltetrahydrofolate. K562.4CF cells exhibited elevated methotrexate uptake relative to parental cells, attributable to a 10-fold increased influx Vmax. The rate of methotrexate efflux in K562.4CF cells was somewhat increased (55%) as well. The transport system in K562.4CF cells had similar and high apparent binding affinities for methotrexate and 5-formyltetrahydrofolate and a markedly reduced affinity for folic acid, properties typically associated with the "classical" methotrexate/tetrahydrofolate cofactor transporter in tumor cells. Methotrexate uptake in K562.4CF cells decreased substantially under nonselective conditions; high levels of transport were restored in 0.4 nM 5-formyltetrahydrofolate. Treatment of parental and K562.4CF cells with N-hydroxysuccinimide methotrexate inhibited methotrexate influx. N-Hydroxysuccinimide-[3H]methotrexate (700 nM) radiolabeled a broadly migrating band at Mr 76,000-85,000. Incorporation from N-hydroxysuccinimide-[3H]methotrexate into this band was increased 7-fold in K562.4CF over parental cells and was blocked by unlabeled methotrexate, (6S)-5-formyltetrahydrofolate, or, to a lesser extent, folic acid. Whereas incubation with endoglycosidase F had no effect on the electrophoretic migration of the labeled protein, treatment with endoglycosidase F and glycopeptidase F, or endo-beta-galactosidase, reduced the apparent molecular weight to Mr approximately 52,000 or approximately 58,000, respectively. These results suggest that the high-affinity transporter in K562.4CF cells is an N-linked glycoprotein containing internal beta-galactosidic linkages in, or immediately after, unbranched poly-N-acetyllactosamine sequences. Differences in the level of glycosylation may, in part, account for the disparity in the apparent sizes of the homologous folate transport proteins from human and murine cells.
选择一株K562人红白血病细胞系（命名为K562.4CF），使其在生长限制浓度（0.4 nM）的（6R,S）-5-甲酰四氢叶酸中四氢叶酸辅因子转运增加。与亲代细胞相比，K562.4CF细胞的甲氨蝶呤摄取量升高，这归因于流入Vmax增加了10倍。K562.4CF细胞中甲氨蝶呤的外排速率也有所增加（55%）。K562.4CF细胞中的转运系统对甲氨蝶呤和5-甲酰四氢叶酸具有相似且较高的表观结合亲和力，而对叶酸的亲和力明显降低，这些特性通常与肿瘤细胞中的“经典”甲氨蝶呤/四氢叶酸辅因子转运体相关。在非选择性条件下，K562.4CF细胞中甲氨蝶呤的摄取量大幅下降；在0.4 nM 5-甲酰四氢叶酸中可恢复高水平的转运。用N-羟基琥珀酰亚胺甲氨蝶呤处理亲代细胞和K562.4CF细胞可抑制甲氨蝶呤的流入。N-羟基琥珀酰亚胺-[3H]甲氨蝶呤（700 nM）标记了一条迁移范围较宽的条带，其分子量为76,000 - 85,000。与亲代细胞相比，K562.4CF细胞中从N-羟基琥珀酰亚胺-[3H]甲氨蝶呤掺入该条带的量增加了7倍，并且被未标记的甲氨蝶呤、（6S）-5-甲酰四氢叶酸或程度较轻的叶酸所阻断。虽然用内切糖苷酶F孵育对标记蛋白的电泳迁移没有影响，但用内切糖苷酶F和糖肽酶F或内切β-半乳糖苷酶处理后，表观分子量分别降至约52,000或约58,000。这些结果表明，K562.4CF细胞中的高亲和力转运体是一种N-连接糖蛋白，在未分支的多聚N-乙酰乳糖胺序列中或之后含有内部β-半乳糖苷键。糖基化水平的差异可能部分解释了人和鼠细胞中同源叶酸转运蛋白表观大小的差异。
