Okajima T, Goto S, Tanizawa K, Tagaya M, Fukui T, Shimofuruya H, Suzuki J
Faculty of Agriculture, Kinki University, Nara.
J Biochem. 1995 May;117(5):980-6. doi: 10.1093/oxfordjournals.jbchem.a124830.
A cDNA encoding porcine brain UMP-CMP kinase has been isolated using two oligonucleotide probes synthesized on the basis of the partial amino acid sequences of the purified enzyme. The isolated cDNA consisted of 1,626 nucleotides including the coding region for a polypeptide of 196 amino acid residues with a calculated molecular weight of 22,279. The enzyme showed an overall sequence identity of about 40 and 50%, respectively, with adenylate kinases from mammalian muscle and Escherichia coli and UMP-CMP kinases from Saccharomyces cerevisiae and Dictyostelium discoideum. The two highly conserved residues, Thr-39 and Leu-66, in adenylate kinases, which are located close to the adenine ring of the bound AMP, are replaced by Ala and Ile, respectively, at the corresponding positions in UMP-CMP kinases. The entire structural gene was inserted 3'-downstream of the strong promoter in the expression plasmid pET-3b. E. coli BL21(DE3) cells carrying the resultant plasmid produced the active enzyme in a soluble state, most efficiently upon induction at 37 degrees C with 0.02 mM isopropyl-beta-D-thiogalactoside. The purified recombinant enzyme catalyzed specific phosphoryl transfer from ATP to UMP and CMP.
利用根据纯化酶的部分氨基酸序列合成的两个寡核苷酸探针，分离出了编码猪脑UMP-CMP激酶的cDNA。分离出的cDNA由1626个核苷酸组成，包括一个编码196个氨基酸残基多肽的编码区，计算分子量为22279。该酶与哺乳动物肌肉和大肠杆菌的腺苷酸激酶以及酿酒酵母和盘基网柄菌的UMP-CMP激酶的总体序列同一性分别约为40%和50%。腺苷酸激酶中靠近结合的AMP腺嘌呤环的两个高度保守残基Thr-39和Leu-66，在UMP-CMP激酶的相应位置分别被Ala和Ile取代。将整个结构基因插入表达质粒pET-3b中强启动子的3'下游。携带所得质粒的大肠杆菌BL21(DE3)细胞以可溶状态产生活性酶，在37℃用0.02 mM异丙基-β-D-硫代半乳糖苷诱导时效率最高。纯化的重组酶催化从ATP到UMP和CMP的特异性磷酸转移。
