文献类型：期刊文献

中文题名：重组酿酒酵母腺苷激酶的表达、纯化和鉴定(英文)

英文题名：Expression,Purification,and Characterization of Recombinant Saccharomyces cerevisiae Adenosine Kinase

作者：吕晓冰[1];吴海珍[1];叶江[1];范怡[1];张惠展[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2003

卷号：35

期号：7

起止页码：666

中文期刊名：生物化学与生物物理学报

收录：CSTPCD;;Scopus;北大核心:【北大核心2000】；CSCD:【CSCD2011_2012】；PubMed;

基金：广东肇庆星湖股份有限公司基金资助~~

语种：中文

中文关键词：腺苷;ATP;表达;细胞;纯化;ET;克隆;激酶;酿酒酵母;重组蛋白质

外文关键词：adenosine kinase; protein purification; enzyme kinetics; Saccharomyces cerevisiae

摘要：腺苷激酶 (adenosinekinase ,AK)是控制细胞中腺苷浓度的一种关键酶 ,在许多细胞和组织中发挥着重要的生理效应子作用。从酿酒酵母 (Saccharomycescerevisiae)中克隆了腺苷激酶基因 (ak) ,并将其接入大肠杆菌pET16b表达质粒中进行表达。重组蛋白质经部分纯化后 ,测定其酶动力学常数 ,结果表明该酶对腺苷的Km 值为 (3.5± 0 .2 ) μmol L ,对ATP的Km 值为(10 0 .0± 11.0 ) μmol L ,对腺苷的kcat值为 (15 30± 2 0 )min- 1 ,对ATP的kcat值为 (14 4 8± 2 5 )min- 1 。该酶对其他核苷和脱氧核苷的Km 值测定数据表明 ,从酵母中克隆得到的该重组腺苷激酶具有较高的底物特异性。
Adenosine kinase (AK), a key enzyme in the regulation of the cellular concentrations of adenosine (A), is an important physiological effector of many cells and tissues. In this article, we reported that ak , which encoded adenosine kinase, was cloned from Saccharomyces cerevisiae , sequenced, and overexpressed in E. coli using the pET16b expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of S. cerevisiae AK revealed K m values of (3.5±0.2) μmol/L for adenosine and (100.0±11.0) μmol/L for ATP, with k cat of (1530±20) min -1 for adenosine and (1448±25) min -1 for ATP. The determination of the K m value for other nucleosides and deoxynucleoside indicated that the nucleoside specificity of this enzyme from yeast was quite high.