Creatine kinase protein sequence encoded by a cDNA made from Torpedo californica electric organ mRNA.
Creatine kinase protein sequence encoded by a cDNA made from Torpedo californica electric organ mRNA.
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肌酸激酶蛋白序列由加州鱼雷电器官 mRNA 制成的 cDNA 编码。
DOI：
10.1073/pnas.81.22.7007
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发表时间：
1984
影响因子：
11.1
通讯作者：
Baxter,JD
中科院分区：
文献类型：
--
作者：
West,BL;Babbitt,PC;Mendez,B;Baxter,JD
肌酸激酶（ATP肌酸n -磷酸转移酶，EC 2.7.3.2）在维持高能量需求组织（如肌肉和大脑）的ATP水平方面很重要。要完全了解它的功能，就需要了解它的氨基酸序列。为了获得编码肌酸激酶序列的cDNA克隆，利用加利福尼亚鱼雷的电器官mRNA构建cDNA库，并通过电器官与肝脏来源的32p标记cDNA的差异集落杂交进行筛选。克隆的dna已经被分离出来，可以在体外翻译电器官mRNA后大量合成40,000-43,000种物质。其中一个克隆ck5278通过双脱氧M13方法测序，发现其编码Mr 42,941蛋白，该蛋白与兔肌肌酸激酶的已知部分序列同源68%，其组成与鸡和兔组织的肌酸激酶相似。相比之下，与使用其他底物的已知激酶序列没有明显的同源性。RNA印迹杂交分析表明ck5288与一个1600碱基对的mRNA互补。引物延伸分析表明，ck5288只比全长cDNA短5个核苷酸，这意味着它编码了一个完整的蛋白序列。这一完整序列的可用性将有助于利用位点特异性诱变等技术进一步研究肌酸激酶的结构和功能。
Creatine kinase (ATP creatine N-phosphotransferase, EC 2.7.3.2) is important in the maintenance of ATP levels in high energy-requiring tissues such as muscle and brain. A complete understanding of its function requires knowledge of its amino acid sequence. To obtain cDNA clones encoding creatine kinase sequences, a cDNA bank was constructed using mRNA from the electric organ of Torpedo californica and was screened by comparing differential colony hybridization of electric organ and liver-derived 32P-labeled cDNAs. Cloned DNAs have been isolated that can arrest the abundant synthesis of Mr 40,000-43,000 material seen after in vitro translation of electric organ mRNA. One of the clones, CK52g8, was sequenced by the dideoxy M13 method and was found to encode a Mr 42,941 protein, which is 68% homologous to a known partial sequence of rabbit muscle creatine kinase and which has a composition similar to creatine kinases from chicken and rabbit tissues. By contrast, no significant homology was found with the known sequences of kinases that use other substrates. RNA blot hybridization analysis indicated that CK52g8 is complementary to a 1600-base-pair mRNA. Primer extension analysis indicated that CK52g8 is only 5 nucleotides short of a full-length cDNA, implying that it encodes a complete protein sequence. The availability of this complete sequence should be useful in further studies of creatine kinase structure and function using techniques such as site-specific mutagenesis.