重组PICK1蛋白的多聚形式及其与Ca^(2+)和Mg^(2+)的结合  

Oligomeric Properties of Recombinant PICK1 and Its Binding Capacity with Ca^(2+) and Mg^(2+)

作　　者：石亚伟[1] 张蕾[1] 肖虹[2] 王丽丽[1] 

机构地区：[1]教育部化学生物学与分子工程重点实验室山西大学生物技术研究所,太原030006 [2]山西医科大学第一附属医院病理科,太原030006

出　　处：《中国生物化学与分子生物学报》2007年第12期1012-1018,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然基金项目资助(No.30400065);山西省自然基金资助(No.2007021033);山西省留学基金项目~~

摘　　要：蛋白激酶Cα相互作用蛋白1(PICK1)是从线虫到人的所有生物中非常保守的一类存在于细胞质中的膜结合蛋白,在蛋白质转运,以及细胞内信号转导过程中发挥重要作用.通过基因重组技术获得PICK1及其截短的N-PDZ(1~110残基)和BAR-C(128~416残基)重组蛋白,结合变性与非变性聚丙烯酰胺凝胶电泳,以及分子排阻层析,表明溶液中的PICK1主要以二聚体形式存在.利用荧光光谱分析PICK1与金属离子Ca2+和Mg2+的结合情况.结果表明,在0.02 mol/L Hepes,pH 7.2,随着2种金属离子的不断滴加,PICK1在338 nm处的最大荧光强度逐渐降低,PICK1与Ca2+结合常数为Ka1=(2.34±0.20)×106L.mol-1,Ka2=(7.75±0.62)×105L.mol-1,而Mg2+结合常数为Ka=(5.00±0.40)×106L.mol-1.另外,对PICK1的N端区域N-PDZ和C端区域BAR-C的重组片段与金属离子Ca2+和Mg2+结合情况进一步分析表明,Ca2+既能与PICK1的N端N-PDZ结合,又可与C端BAR-C结合,而Mg2+只结合在PICK1的N-PDZ区域.比较Ca2+或Mg2+对PICK1结合脂质的影响,显示Ca2+能明显增强蛋白和脂质的结合.Protein interacting with Cα kinase 1 （PICK1）, an evolutionaryly conserved protein widely present in creatures from C. elegans to human being, is a peripheral membrane protein appeared in the cytoplasm and plays an important role in the process of protein translocation, and the intracellular signal transduction. PICK1 and its truncated fragments （N-PDZ and BAR-C） were expressed in E. coli BL21 （DE3） and purified by affinity chromatography and gel filtration. PICK1 was in the form of dimer in solution by the analysis of non- denaturing PAGE and molecular exclusion chromatography . The binding properties of PICK1 with Ca^2＋ and Mg^2＋ was examined by fluorescence spectra. Ca^2＋ and Mg^2＋ have the similar affinity with PICK1 at 2：1 stoichiometry in 0.02 mol ·L^-1 Hepes, pH 7.4 at 25 ℃. The conditional binding constants of complex PICK1- Ca^2＋ and PICK1- Mg^2＋ are Ka1 = （2.34±0.20） × 10^6 L·mol^-1 ,Ka2= （7.75 ±0.62） × 10^5 L·mol^-1 and Ka ： （5.00 ± 0.40） × 10^6 L·mol^-1, respectively. Moreover, Ca^2＋ can bind to both N-PDZ and BAR-C regions of PICK1, while Mg^2＋ to N-PDZ region only. Compared to Mg^2＋ , Ca^2＋ significantly enhances the lipid binding of PICK1 according to the fluorescence titration assays.

关 键 词：蛋白激酶Cα相互作用蛋白1 二聚体 钙离子 镁离子 荧光光谱 

分 类 号：Q51[生物学—生物化学]