Miura K, Kurosawa Y, Kanai Y
Institute for Comprehensive Medical Science, Fujita Health University, Aichi, Japan.
Biochem Biophys Res Commun. 1994 Mar 30;199(3):1388-93. doi: 10.1006/bbrc.1994.1384.
In a previous paper (Biochem. Biophys. Res. Commun. 187, 375-380, 1992), we reported cloning of a gene for nucleobindin (Nuc) from mouse and human. Nuc was identified as a secreted protein with DNA-binding properties. We noticed that Nuc includes two units of sequence similar to the EF hand motif, a calcium-binding motif, between a region rich in basic amino acids, which is presumably a DNA-binding site, and a leucine zipper which is a dimerization motif. In the present study, we examined the calcium-binding activity of Nuc. Mouse and human recombinant Nuc (rNuc), as well as two mutant rNuc proteins, were synthesized in Escherichia coli. Mouse and human rNuc and one mutant rNuc, in which the region from the second EF hand motif to the C-terminus had been deleted, all had calcium-binding activity. By contrast, another mutant rNuc in which both EF hand motifs had been deleted lacked this activity. Analysis by circular dichroism spectrometry indicated that addition of Ca2+ ions induces a structural change in Nuc, presumably an increase in the amount of alpha helix. However, addition of Ca2+ ions did not seem to cause any change in DNA-binding activity.
在之前的一篇论文（《生物化学与生物物理研究通讯》187, 375 - 380, 1992）中，我们报道了从小鼠和人类中克隆出核结合蛋白（Nuc）的基因。Nuc被鉴定为一种具有DNA结合特性的分泌蛋白。我们注意到Nuc包含两个类似于EF手基序（一种钙结合基序）的序列单元，位于富含碱性氨基酸的区域（推测为DNA结合位点）和亮氨酸拉链（一种二聚化基序）之间。在本研究中，我们检测了Nuc的钙结合活性。小鼠和人类重组Nuc（rNuc）以及两种突变rNuc蛋白在大肠杆菌中合成。小鼠和人类rNuc以及一种从第二个EF手基序到C末端区域被删除的突变rNuc都具有钙结合活性。相比之下，另一种两个EF手基序都被删除的突变rNuc则缺乏这种活性。圆二色光谱分析表明，添加Ca2 +离子会诱导Nuc发生结构变化，推测是α螺旋含量增加。然而，添加Ca2 +离子似乎并未引起DNA结合活性的任何变化。
