重组人Pif1的ATP酶活性表征
Characterization of ATPase activity of recombinant human Pif1.
作者信息
Huang Yu, Zhang Deng-Hong, Zhou Jin-Qiu
机构信息
Max-Planck Junior Research Group at the State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
出版信息
Acta Biochim Biophys Sin (Shanghai). 2006 May;38(5):335-41. doi: 10.1111/j.1745-7270.2006.00165.x.
Saccharomyces cerevisiae Pif1p helicase is the founding member of the Pif1 subfamily that is conserved from yeast to human. The potential human homolog of the yeast PIF1 gene has been cloned from the cDNA library of the Hek293 cell line. Here, we described a purification procedure of glutathione S-transferase (GST)-fused N terminal truncated human Pif1 protein (hPif1deltaN) from yeast and characterized the enzymatic kinetics of its ATP hydrolysis activity. The ATPase activity of human Pif1 is dependent on divalent cation, such as Mg2+, Ca2+ and single-stranded DNA. Km for ATP for the ATPase activity is approximately 200 microM. As the ATPase activity is essential for hPif1's helicase activity, these results will facilitate the further investigation on hPif1.
酿酒酵母Pif1p解旋酶是Pif1亚家族的创始成员，该亚家族从酵母到人类都保守存在。酵母PIF1基因潜在的人类同源物已从Hek293细胞系的cDNA文库中克隆出来。在此，我们描述了从酵母中纯化谷胱甘肽S-转移酶（GST）融合的N端截短型人类Pif1蛋白（hPif1deltaN）的方法，并对其ATP水解活性的酶动力学进行了表征。人类Pif1的ATP酶活性依赖于二价阳离子，如Mg2+、Ca2+和单链DNA。ATP酶活性的ATP的Km约为200 microM。由于ATP酶活性对hPif1的解旋酶活性至关重要，这些结果将有助于对hPif1的进一步研究。
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