Yamaoka T, Yano M, Kondo M, Sasaki H, Hino S, Katashima R, Moritani M, Itakura M
Division of Genetic Information, Institute for Genome Research, Department of Clinical and Molecular Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan.
J Biol Chem. 2001 Jun 15;276(24):21285-91. doi: 10.1074/jbc.M011103200. Epub 2001 Apr 4.
To clarify the contributions of amidophosphoribosyltransferase (ATase) and its feedback regulation to the rates of purine de novo synthesis, DNA synthesis, protein synthesis, and cell growth, mutated human ATase (mhATase) resistant to feedback inhibition by purine ribonucleotides was engineered by site-directed mutagenesis and expressed in CHO ade (-)A cells (an ATase-deficient cell line of Chinese hamster ovary fibroblasts) and in transgenic mice (mhATase-Tg mice). In Chinese hamster ovary transfectants with mhATase, the following parameters were examined: ATase activity and its subunit structure, the metabolic rates of de novo and salvage pathways, DNA and protein synthesis rates, and the rate of cell growth. In mhATase-Tg mice, ATase activity in the liver and spleen, the metabolic rate of the de novo pathway in the liver, serum uric acid concentration, urinary excretion of purine derivatives, and T lymphocyte proliferation by phytohemagglutinin were examined. We concluded the following. 1) ATase and its feedback inhibition regulate not only the rate of purine de novo synthesis but also DNA and protein synthesis rates and the rate of cell growth in cultured fibroblasts. 2) Suppression of the de novo pathway by the salvage pathway is mainly due to the feedback inhibition of ATase by purine ribonucleotides produced via the salvage pathway, whereas the suppression of the salvage pathway by the de novo pathway is due to consumption of 5-phosphoribosyl 1-pyrophosphate by the de novo pathway. 3) The feedback inhibition of ATase is more important for the regulation of the de novo pathway than that of 5-phosphoribosyl 1-pyrophosphate synthetase. 4) ATase superactivity leads to hyperuricemia and an increased bromodeoxyuridine incorporation in T lymphocytes stimulated by phytohemagglutinin.
为阐明氨甲酰磷酸核糖转移酶（ATase）及其反馈调节对嘌呤从头合成、DNA合成、蛋白质合成和细胞生长速率的贡献，通过定点诱变构建了对嘌呤核糖核苷酸反馈抑制具有抗性的突变型人ATase（mhATase），并在CHO ade(-)A细胞（中国仓鼠卵巢成纤维细胞的ATase缺陷细胞系）和转基因小鼠（mhATase-Tg小鼠）中表达。在转染了mhATase的中国仓鼠卵巢细胞中，检测了以下参数：ATase活性及其亚基结构、从头合成途径和补救途径的代谢速率、DNA和蛋白质合成速率以及细胞生长速率。在mhATase-Tg小鼠中，检测了肝脏和脾脏中的ATase活性、肝脏中从头合成途径的代谢速率、血清尿酸浓度、嘌呤衍生物的尿排泄以及植物血凝素刺激的T淋巴细胞增殖。我们得出以下结论。1）ATase及其反馈抑制不仅调节培养的成纤维细胞中嘌呤从头合成的速率，还调节DNA和蛋白质合成速率以及细胞生长速率。2）补救途径对从头合成途径的抑制主要是由于通过补救途径产生的嘌呤核糖核苷酸对ATase的反馈抑制，而从头合成途径对补救途径的抑制是由于从头合成途径消耗了5-磷酸核糖-1-焦磷酸。3）ATase的反馈抑制对从头合成途径的调节比5-磷酸核糖-1-焦磷酸合成酶的反馈抑制更重要。4）ATase超活性导致高尿酸血症，并增加植物血凝素刺激的T淋巴细胞中溴脱氧尿苷的掺入。
