Niemann G, von Besser H, Walter R D
Bemhard Nocht Institute for Tropical Medicine, Department of Biochemistry, Hamburg, Federal Republic of Germany.
Biochem J. 1996 Jul 1;317 ( Pt 1)(Pt 1):135-40. doi: 10.1042/bj3170135.
A southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp. In the 5' non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified. The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni(2+)-chelating affinity chromatography. The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa. On the basis of gel filtration analyses it is shown that the active enzyme is a dimer. The specific enzyme activity was determined to be 4.2 mumol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity. The Km value for L-ornithine was determined as 44 microM. The Ki values for putrescine, alpha-diffluoromethylornithine, alpha-hydrazino-ornithine and alpha-methylornithine were calculated as 51, 34, 0.34 and 42 microM respectively.
对泛裸口线虫鸟氨酸脱羧酶（ODC）基因的Southern印迹分析表明，它是一个单拷贝基因，位于基因组3.2 kb的EcoRI片段上。从基因组EMBL-4文库中分离并纯化了具有ODC基因序列的噬菌体克隆。分析噬菌体DNA插入片段，分离出一个包含完整ODC基因的3.2 kb EcoRI片段。该片段的核苷酸序列分析表明，该基因被两个分别为47 bp和49 bp的内含子打断。在该基因的5'非翻译区，鉴定出了推定的AP1、VPE2和c-Myc结合位点。ODC cDNA在细菌系统中表达为His融合蛋白，并通过Ni(2+)-螯合亲和层析纯化该酶。根据cDNA推导并经SDS/PAGE显示，亚基分子量为47.1 kDa。凝胶过滤分析表明，活性酶是一种二聚体。测定的比酶活性为4.2 μmol CO2/分钟/毫克蛋白。该酶依赖磷酸吡哆醛作为辅因子，二硫赤藓糖醇或其他巯基还原剂的存在对最大活性至关重要。L-鸟氨酸的Km值测定为44 μM。腐胺、α-二氟甲基鸟氨酸、α-肼基鸟氨酸和α-甲基鸟氨酸的Ki值分别计算为51、34、0.34和42 μM。
