Kan J L, Jannatipour M, Taylor S M, Moran R G
Department of Biochemistry, University of Southern California, Los Angeles 90033.
Gene. 1993 Dec 31;137(2):195-202.
Three of the enzymatic activities of de novo purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS) and glycinamide ribonucleotide formyltransferase (GART), can be catalyzed by a single 110-kDa protein in mouse cells. Western blots using a polyclonal antibody (Ab) to this protein identified two species, the trifunctional 110-kDa protein and a 50-kDa cytosolic protein with GARS, but not GART activity. We used Ab and, subsequently, oligodeoxyribonucleotide screens to isolate cDNAs corresponding to these two proteins from mouse T-cell cDNA expression libraries. The sequence of one class of these cDNAs and the partial sequence of a corresponding genomic clone defined an open reading frame (ORF) encoding a 1010-amino-acid (aa) protein, individual domains of which showed high homology to each of the monofunctional bacterial GARS, AIRS and GART proteins, and to each domain of chicken and human trifunctional GARS-AIRS-GARTs. cDNAs corresponding to the smaller protein contained a 1.3-kb ORF with complete identity to the GARS domain of, but with a 3' untranslated region different from, the trifunctional cDNAs. Hence, both cDNAs appear to derive from the same gene due to either differential splicing or use of an intronic polyadenylation signal. The functional requirement for the expression of both trifunctional protein with GARS activity and monofunctional, catalytically active GARS is unknown.
在小鼠细胞中，从头合成嘌呤的三种酶活性，即甘氨酰胺核糖核苷酸合成酶（GARS）、氨基咪唑核糖核苷酸合成酶（AIRS）和甘氨酰胺核糖核苷酸甲酰基转移酶（GART），可由一种单一的110 kDa蛋白质催化。使用针对该蛋白质的多克隆抗体（Ab）进行的蛋白质免疫印迹法鉴定出两种蛋白，即具有三功能的110 kDa蛋白质和一种具有GARS活性但不具有GART活性的50 kDa胞质蛋白。我们使用抗体，随后又通过寡聚脱氧核糖核苷酸筛选，从小鼠T细胞cDNA表达文库中分离出与这两种蛋白质相对应的cDNA。这些cDNA中的一类序列以及相应基因组克隆的部分序列确定了一个开放阅读框（ORF），其编码一种1010个氨基酸（aa）的蛋白质，该蛋白质的各个结构域与单功能细菌GARS、AIRS和GART蛋白质以及鸡和人三功能GARS - AIRS - GART的每个结构域都具有高度同源性。与较小蛋白质相对应的cDNA包含一个1.3 kb的ORF，与三功能cDNA的GARS结构域完全相同，但3'非翻译区不同。因此，由于差异剪接或使用内含子聚腺苷酸化信号，这两种cDNA似乎都来自同一个基因。具有GARS活性的三功能蛋白质和单功能、具有催化活性的GARS表达的功能需求尚不清楚。
