Xiao-Bing Lu, Hai-Zhen Wu, Jiang Ye, Yi Fan, Hui-Zhan Zhang
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引用次数: 0
摘要
腺苷激酶(Adenosine kinase, AK)是调控细胞内腺苷(Adenosine, a)浓度的关键酶,是许多细胞和组织的重要生理效应因子。在本文中,我们从酿酒酵母中克隆了编码腺苷激酶的ak蛋白,对其进行测序,利用pET16b表达系统在大肠杆菌中过表达,并利用常规蛋白纯化技术纯化重组蛋白,使其具有明显的均匀性。对酿酒酵母AK的动力学分析表明,其腺苷K(m)值为(3.5+/-0.2)微mol/L, ATP K(m)值为(100.0+/-11.0)微mol/L,腺苷K(cat)值为(1530+/-20)min(-1), ATP K(cat)值为(1448+/-25)min(-1)。对其他核苷和脱氧核苷的K(m)值测定表明,该酵素对酵母核苷有很高的特异性。
Expression, purification, and characterization of recombinant Saccharomyces cerevisiae adenosine kinase.
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) micromol/L for adenosine and (100.0+/-11.0) micromol/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.