Wilson蛋白离子结合功能的研究

Metal binding properties of the Wilson protein

目的 研究Wilson蛋白 (ATP7B酶 )的离子结合功能 ,探讨锌剂治疗Wilson病的机制。方法 应用逆转录 聚合酶链反应 (RT PCR)扩增目的基因 ,GST基因融合载体表达并纯化融合蛋白 ,凝血酶酶切并收集目的蛋白 ,应用离子螯合层析和放射性锌印迹技术研究蛋白不同离子的结合能力。结果 ATP7B的离子结合能力为Cu(Ⅰ ) >Zn(Ⅱ ) >Ni(Ⅱ ) >Cu(Ⅱ ) ,并发现锌离子和铜离子对蛋白的结合具有竞争作用。结论 本实验证明了ATP7B以一价铜离子的形式行使其运铜功能 ,ATP7B不仅有结合铜离子的功能 ,而且有结合其他离子的功能 ;铜锌两种金属离子在蛋白结合位点的竞争作用可能是锌剂治疗WD的机制之一。

Objective To investigate the copper transporting ability of Wilson protein, and the mechanism of zinc therapy. Methods Using RT-PCR to amplify a 1.95 kb copper binding domain fragment of Wilson protein (WCBD) and cloning it to pGEX-4T-1, the GST-WCBD was expressed in BL-21 under the IPTG induction. Purified the GST-WCBD with GST-columns, cleaved the GST part with Thrombin and acquired the WCBD. The metal binding properties to Cu(Ⅱ), Zn(Ⅱ), Fe(Ⅲ), Ni(Ⅱ), Ag(Ⅰ) with IMAC and 65 Zn blotting analysis were carried out. Results The WCBD was found having varying affinities with different metals as follow: Cu(Ⅰ)> Zn(Ⅱ) >Ni(Ⅱ) >Cu(Ⅱ). Zn(Ⅱ) was discovered able to compete with Cu(Ⅰ) in the binding domain. Conclusion Wilson protein delivering copper as Cu(Ⅰ) was suggested to be demonstrated. The capacity of zinc to compete with copper may be a mechanism in zinc treatment to this disease.