Identification, cloning and expression of the mouse N-acetylglutamate synthase gene
Identification, cloning and expression of the mouse N-acetylglutamate synthase gene
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DOI：
10.1042/bj20020161
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发表时间：
2002-06-15
影响因子：
4.1
通讯作者：
Tuchman, M
中科院分区：
文献类型：
--
作者：
Caldovic, L;Morizono, H;Tuchman, M
在排尿素动物中，N-乙酰谷氨酸（NAG）是氨甲酰磷酸合成酶I（CPSI）的必需变构激活剂，CPSI是尿素循环中的第一种酶，NAG合成酶（NAGS; EC 2.3.1.1）催化肝脏和肠线粒体中谷氨酸和乙酰辅酶A形成NAG。这种酶被认为通过产生不同量的NAG来调节尿素生成，从而调节CPSI活性。此外，NAGS的遗传缺陷与高氨血症有关，可能是由于CPSI活性的丧失。虽然NAGS蛋白在哺乳动物中的存在已经被发现了几十年，但该基因仍然难以捉摸。我们确定了小鼠（小家鼠）和人类NAGS基因使用它们的相似性，各自的粗糙脉孢菌基因。从小鼠肝脏cDNA文库中克隆了NAGS基因，发现其编码一个2.3kb的信息，在肝脏和小肠中高表达，在肾脏、脾脏和睾丸中表达水平较低。推导的氨基酸序列在N-末端含有推定的线粒体靶向信号。cDNA序列补充argA（NAGS）缺陷型大肠杆菌菌株，逆转其精氨酸营养缺陷型。His标记的前体蛋白和两个推定的成熟蛋白在E.大肠杆菌，并通过使用镍亲和柱纯化至表观均一性。前体蛋白和两个推定的成熟蛋白催化NAGS反应，但其中一个推定的成熟酶的活性显着高于前体蛋白。L-精氨酸的加入使纯化的重组NAGS酶的催化活性增加约10%。2-6倍。
In ureotelic animals, N-acetylglutamate (NAG) is an essential allosteric activator of carbamyl phosphate synthetase I (CPSI), the first enzyme in the urea cycle, NAG synthase (NAGS; EC 2.3.1.1) catalyses the formation of NAG from glutamate and acetyl-CoA in liver and intestinal mitochondria. This enzyme is supposed to regulate ureagenesis by producing variable amounts of NAG, thus modulating CPSI activity. Moreover, inherited deficiencies in NAGS have been associated with hyperamtnonaemia, probably due to the loss of CPSI activity. Although the existence of the NAGS protein in mammals has been known for decades, the gene has remained elusive. We identified the mouse (Mus musculus) and human NAGS genes using their similarity to the respective Neurospora crassa gene. NAGS was cloned from a mouse liver cDNA library and was found to encode a 2.3 kb message, highly expressed in liver and small intestine with lower expression levels in kidney, spleen and testis. The deduced amino acid sequence contains a putative mitochondrial targeting signal at the N-terminus. The cDNA sequence complements an argA (NAGS)-deficient Escherichia coli strain, reversing its arginine auxotrophy. His-tagged versions of the pre-protein and two putative mature proteins were each overexpressed in E. coli, and purified to apparent homogeneity by using a nickel-affinity column. The pre-protein and the two putative mature proteins catalysed the NAGS reaction but one of the putative mature enzymes had significantly higher activity than the pre-protein. The addition Of L-arginine increased the catalytic activity of the purified recombinant NAGS enzymes by approx. 2-6-fold.