Abstract Thymidylate kinase (TMPK, EC2.7.4.9) is the enzyme that converts deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP) in the synthesis of dTTP, an essential building block of DNA. To date, there is only one gene (TYMK) known to encode TMPK in mammalian cells. In this study, we investigated the distribution of TMPK activity and protein in subcellular fractions by using activity measurements and by using a specific antibody against TYMK-encoded TMPK (canonical TMPK). TMPK activity was detected in all subcellular fractions, of which the mitochondrial outer membrane contained the highest activity. High levels of canonical TMPK protein were detected in the cytosolic fraction, whereas low levels were found in the nuclear and mitochondrial matrix fractions. Strikingly, despite the detection of high TMPK activity in the mitochondrial outer membrane, canonical TMPK protein was not detected in this fraction. These results suggest that the TMPK activity detected in the outer membrane fraction may originate from a novel dTMP kinase, distinct from the canonical TYMK.

中文翻译：

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新型胸苷酸激酶活性的鉴定

摘要 胸苷酸激酶 (TMPK, EC2.7.4.9) 是在 dTTP 合成过程中将脱氧胸苷单磷酸 (dTMP) 转化为脱氧胸苷二磷酸 (dTDP) 的酶，dTTP 是 DNA 的基本组成部分。迄今为止，已知只有一种基因 (TYMK) 在哺乳动物细胞中编码 TMPK。在这项研究中，我们通过使用活性测量和使用针对 TYMK 编码的 TMPK（规范 TMPK）的特异性抗体，研究了亚细胞组分中 TMPK 活性和蛋白质的分布。在所有亚细胞组分中均检测到 TMPK 活性，其中线粒体外膜的活性最高。在细胞质部分中检测到高水平的典型 TMPK 蛋白，而在核和线粒体基质部分中发现低水平。引人注目的是，尽管在线粒体外膜中检测到高 TMPK 活性，但在该部分中未检测到典型的 TMPK 蛋白。这些结果表明在外膜组分中检测到的 TMPK 活性可能源自一种新的 dTMP 激酶，不同于经典的 TYMK。