关键词: 信号分子;鸟苷酸类;PH结构域;蛋白激酶C;活性

    摘 要:目的 鉴定表达guanine-nucleotide-releasing factor(GRF)的pleckstrin homology(PH)结构域与谷胱甘肽转移酶(GST)融合蛋白的可溶性，并进一步检测该重组蛋白与蛋白激酶C(PKC)的结合及对PKC活性的影响. 方法 将表达GRF的PH结构域与GST融合蛋白的菌体经超声裂解，上清中的融合蛋白用琼脂糖珠锚定纯化.Western blot检测融合蛋白与PKC的结合，以PKC活性检测试剂盒测定结合后PKC的活性变化. 结果 获得信号分子GRF的PH结构域-GST融合蛋白的可溶性表达.Western blot结果表明，GRF的PH结构域可在体外与PKC结合.PKC活性实验显示，GRF的PH结构域抑制PKC活性. 结论 GRF的PH结构域可在体外与PKC结合并对PKC的活性具有下调作用.

    Recombinant GRF PH domain down┐regulates activ┐ity of protein kinase C

    JI Shao-Ping，YAO Li-Bo，HAN Yue-Heng，LIU Xin-Ping，SU Cheng-Zhi

    Department of Biochemistry and Molecular Biology，Faculty of Preclinical Medicine，Fourth Military Medical University，Xi'an710033，China

    Keywords:signaling molecule;guanine-nucleotide-releasing factor for Ras;PH domain;protein kinase C;ac-tivity

    Abstract:AIM To investigate association of guanine-nu-cleotide-releasing factor for Ras(GRF)PH domain and pro-tein kinase C(PKC)and to examine effect of GRF PH do-main on activity of protein kinase C.METHODS GRF PH domain-GST fusion protein was expressed in E.coli and puri-fied by glutathione agarose beads.The expressed fusion pro-Tel.(029)3374516 Email.jishaoping@hotmail.com tein was harvested by lysing the E.coli with ultrasonic.After centrifugation ......