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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation.
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Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation.

机译：来自肺腺癌细胞的硒磷酸合成酶基因：用于回收L-硒代半胱氨酸的Sps1和用于亚硒酸盐同化的Sps2。

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摘要

A labile selenium donor compound monoselenophosphate is synthesized from selenide and ATP by selenophosphate synthetase (SPS). In the present study, Sps1 and Sps2 were cloned from a cDNA library prepared from human lung adenocarcinoma cells (NCIH441). The human lung Sps1 has been cloned as an ORF of 1,179 bp, identical in sequence to that of the recently revised human liver Sps1. The in-frame TGA codon of the lung Sps2 was genetically altered to TGT (Cys) to obtain the Sps2Cys gene. Expression of the recombinant plasmids containing Sps1 or Sps2Cys was highly toxic to Escherichia coli host cells grown aerobically. Accordingly, the human lung Sps homologs were characterized by an in vivo complementation assay using a selD mutant strain. An added selenium source and a low salt concentration (0.1-0.25% NaCl) in the medium were required for reproducible and sensitive in vivo complementation. Sps2Cys effectively complemented the selD mutant, and the resulting formate dehydrogenase H activity was as high as that of WT E. coli MC4100. In contrast, only a weak complementation of the selD mutant by the Sps1 gene was observed when cells were grown in selenite media. Better complementation with added l-selenocysteine suggested involvement of a selenocysteine lyase for mobilization of selenium. Based on this apparent substrate specificity of the Sps1 and Sps2 gene products we suggest that the Sps1-encoded enzyme depends on a selenium salvage system that recycles l-selenocysteine, whereas the Sps2 enzyme can function with a selenite assimilation system.
机译：通过硒代磷酸合成酶（SPS）由硒化物和ATP合成不稳定的硒供体化合物单硒代磷酸酯。在本研究中，从人肺腺癌细胞（NCIH441）制备的cDNA文库中克隆了Sps1和Sps2。人肺Sps1已被克隆为1,179 bp的ORF，其序列与最近修订的人肝Sps1的序列相同。将肺中Sps2的框架内TGA密码子遗传修饰为TGT（Cys），以获得Sps2Cys基因。含有Sps1或Sps2Cys的重组质粒的表达对需氧生长的大肠杆菌宿主细胞具有高毒性。因此，通过使用selD突变体菌株的体内互补测定来表征人肺Sps同源物。需要添加硒源和培养基中低盐浓度（0.1-0.25％NaCl）以实现可重现和敏感的体内互补。 Sps2Cys有效地补充了selD突变体，并且所得的甲酸脱氢酶H活性与野生型大肠杆菌MC4100一样高。相反，当细胞在亚硒酸盐培养基中生长时，仅观察到SpS1基因对selD突变体的弱互补。更好的补充l-硒代半胱氨酸的补充表明硒代半胱氨酸裂解酶参与了硒的动员。基于Sps1和Sps2基因产物的这种明显的底物特异性，我们建议Sps1编码的酶取决于回收l-硒代半胱氨酸的硒挽救系统，而Sps2酶可以与亚硒酸盐同化系统一起起作用。

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