氚自杀法筛选中性氨基酸转运缺陷的哺乳动物细胞突变体。
Tritium suicide selection of mammalian cell mutants defective in the transport of neutral amino acids.
作者信息
Finkelstein M C, Slayman C W, Adelberg E A
出版信息
Proc Natl Acad Sci U S A. 1977 Oct;74(10):4549-51. doi: 10.1073/pnas.74.10.4549.
Mouse lymphocytic cells of the established line GF-14 were allowed to accumulate intracellular 3H-labeled aminoisobutyric acid (AIB), frozen, and stored over liquid N2. After internal radiation had reduced survival to 1 in 10(4), survivors were plated and tested for their ability to transport AIB. Out of 200 clones tested, two (designated GF-17 and GF-18) were found to have reductions to 13-35% of the parent in the rate of transport of AIB, L-alanine, L-proline, and L-serine; GF-18 also showed significant reductions in the rate of transport of L-glutamate and DL-cysteine. Little or no change was observed for 10 other amino acids or for thymidine. Kinetic analyses revealed that the mutants were not altered in Km for AIB uptake, but had Vmax values approximately 20% the value of the parent strain, GF-14, suggesting that either the number of AIB transport sites or the turnover rate of the sites has been reduced in the two mutants.
使已建立的GF - 14系小鼠淋巴细胞积累细胞内3H标记的氨基异丁酸（AIB），然后冷冻，并保存在液氮中。在内部辐射使存活率降至万分之一后，对存活细胞进行接种，并测试它们转运AIB的能力。在测试的200个克隆中，发现有两个（命名为GF - 17和GF - 18）在转运AIB、L - 丙氨酸、L - 脯氨酸和L - 丝氨酸的速率方面降至亲本的13% - 35%；GF - 18在转运L - 谷氨酸和DL - 半胱氨酸的速率方面也显著降低。对于其他10种氨基酸或胸苷，未观察到变化或变化很小。动力学分析表明，突变体对AIB摄取的米氏常数（Km）没有改变，但最大反应速度（Vmax）值约为亲本菌株GF - 14的20%，这表明在这两个突变体中，AIB转运位点的数量或位点的周转率有所降低。
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