Suzuki H, Verma D P
Department of Molecular Genetics and The Ohio State Biotechnology Center, Columbus, Ohio 43210-1002.
Plant Physiol. 1991 Feb;95(2):384-9. doi: 10.1104/pp.95.2.384.
A complete nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3) was isolated from a soybean (Glycine max L. var. Prize) nodule cDNA expression library using a previously isolated partial cDNA clone. The N-35 cDNA was expressed in Escherichia coli driven by the lacZ promoter and was found to be functionally active. The uricase activity was detected in the cytoplasmic fraction of E. coli with the same pH optimum and apparent K(m) values as that in the nodules. Because a stop codon is located 15 base pairs upstream of the N-35 initiation codon, it appears that a fusion protein with LacZ was not made, but reinitiation occurred due to the presence of a putative Shine-Dalgarno sequence in the appropriate region. The size of the N-35 polypeptide made in E. coli is identical to that present in soybean nodules and is able to assemble into a tetrameric holoenzyme with the same molecular weight as the native uricase. Thus, the presence of peroxisomes does not appear to be essential for the proper assembly of the holoenzyme in E. coli. These data also indicate that posttranslational modifications or membrane transport are not essential either for the assembly of N-35 into a holoenzyme or for the activity of uricase.
利用先前分离得到的部分cDNA克隆，从大豆（Glycine max L. var. Prize）根瘤cDNA表达文库中分离出一个编码根瘤特异性尿酸酶（EC 1.7.3.3）的完整nodulin-35（N-35）cDNA。N-35 cDNA在lacZ启动子驱动下在大肠杆菌中表达，并被发现具有功能活性。在大肠杆菌的细胞质部分检测到尿酸酶活性，其最适pH值和表观K(m)值与根瘤中的相同。由于一个终止密码子位于N-35起始密码子上游15个碱基对处，似乎没有产生与LacZ的融合蛋白，但由于在适当区域存在一个假定的Shine-Dalgarno序列，发生了重新起始。在大肠杆菌中产生的N-35多肽的大小与大豆根瘤中存在的相同，并且能够组装成与天然尿酸酶分子量相同的四聚体全酶。因此，过氧化物酶体的存在似乎对于全酶在大肠杆菌中的正确组装不是必需的。这些数据还表明，翻译后修饰或膜运输对于N-35组装成全酶或尿酸酶的活性也不是必需的。
