孤立的人类的互补,恢复甲氨蝶呤灵敏度和减少叶酸载体活动甲氨蝶呤transport-defective中国仓鼠卵巢细胞。
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黄SC, Proefke SA Bhushan,马瑟LH
孤立的人类的互补,恢复甲氨蝶呤灵敏度和减少叶酸载体活动甲氨蝶呤transport-defective中国仓鼠卵巢细胞。
J生物化学杂志。1995年7月21日,270 (29):17468 - 75。
- PubMed ID
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7615551 (在PubMed]
- 文摘
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这份报告描述了隔离、核苷酸序列和功能表达人类的互补的恢复减少叶酸载体活动transport-defective细胞。基于同源部分小鼠cDNA探针,两个功能互补隔离从甲氨蝶呤λgt11图书馆准备运输调节K562细胞(K562.4CF)。2.8千碱基(kb)克隆、KS43包含1776碱基对的开放阅读框。2.5 kb克隆,KS32,包含一个内部删除(626个碱基对)缩短开放阅读框和3 '非翻译区。KS43和KS32编码蛋白与多个疏水领域,一个共识N-glycosylation网站,并预测分子质量65和58 kDa,分别。KS43的推导氨基酸序列是老鼠和仓鼠79%和80%的同源序列,分别(迪克逊,k . H。Lanpher, b . C。赵,J。凯利,K。考恩,k . h .(1994)生物。269年化学,17 - 20;威廉姆斯,f·m·R。穆雷r . C。, Underhill, T. M., and Flintoff, W. F. (1994) J. Biol. Chem. 269, 5810-5816). Northern blots identified one primary transcript at 3.1 kb in parental K562, K562.4CF, and transport-impaired K500E cells; transcript levels varied by 7-fold. The expression of both KS43 and KS32 in methotrexate transport-defective Chinese hamster ovary cells restored methotrexate sensitivity and transport. Certain transport characteristics of the transfected cells resembled both the wild type human (K562) and hamster "classical" reduced folate carriers, suggesting the expression of a hybrid system. For instance, based on Ki values, up to a 4-fold increased affinity for 1843U89 over wild type hamster cells (typical of human cells), and a 19-fold increased affinity for methotrexate over K562 cells (typical of hamster cells) was observed. Further, a photoaffinity probe with high specificity for the reduced folate carrier labeled 94-kDa proteins in K562 cells and the transfectant containing the full-length KS43, and a 85-kDa protein in the transfectant containing the 3'-truncated KS32. No specifically labeled proteins were detected in wild type or mock-transfected hamster cells. Collectively, our results suggest that the KS43/KS32 cDNAs encode the human reduced folate carrier; however, additional modulatory/regulatory factors may be required to manifest the full spectrum of transport substrate activities typical of this system.