重组PICK1蛋白的多聚形式及其与Ca2+和Mg2+的结合

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）×10.6 L/mol^-1，Ka2=(7.75±0.62)×10.5 L/mol^-1,而Mg²⁺结合常数为Ka=（5.00±0.40）×10.6 L/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BARC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.

Oligomeric Properties of Recombinant PICK1 and Its Binding Capacity with Ca2+ and Mg2+

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 Ca²⁺ and Mg²⁺ was examined by fluorescence spectra. Ca²⁺ and Mg²⁺ 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 Ca²⁺ and PICK1 Mg²⁺ 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, Ca²⁺ can bind to both N-PDZ and BAR-C regions of PICK1, while Mg²⁺ to N-PDZ region only. Compared to Mg²⁺, Ca²⁺ significantly enhances the lipid binding of PICK1 according to the fluorescence titration assays.