脑红蛋白与Na^+,K^+-ATP酶β2亚基的相互作用及作用位点的鉴定  

Identification of Interaction and Interaction Domains between Neuroglobin and Na^+, K^+-ATPase beta2 Subunit

作　　者：徐文琳[1] 王春丽[1] 张永亮[1] 廖志勇[1] 余利红[1] 孟凡伟[1] 王新兴[2] 尹昭云[2] 钱令嘉[2] 张成岗[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850 [2]军事医学科学院卫生学与环境医学研究所,天津300050

出　　处：《生物化学与生物物理学报》2003年第9期823-828,共6页

基　　金：国家自然科学基金重大项目 (No .90 2 0 80 17);国家高技术研究发展计划资助项目 ( 863计划 ) (No .2 0 0 2AA2 3 40 2 1);海外香港青年学者合作研究基金 (No .3 0 12 80 10 );国家自然科学基金面上项目 (No .3 0 10 0 0 49)联合资助~~

摘　　要：为研究神经系统特异性携氧蛋白———脑红蛋白 (NGB)保护神经元耐受缺氧损伤的分子机制 ,利用酵母双杂交系统从人胎脑cDNA文库中筛选与其有相互作用的蛋白质。序列分析表明 ,其中一个克隆的编码产物与Na+ ,K+ ATP酶 β2亚基 (NKA1b2 )序列一致。随后采用PCR方法从人胎脑cDNA文库中扩增获得NKA1b2全长cDNA。蛋白质结合实验表明 ,原核表达的NGB与体外转录翻译得到的NKA1b2在细胞外有结合作用。免疫共沉淀实验证明二者在生理条件下能够以复合物的形式存在。利用NGB系列短截体研究相互作用的位点发现 ,NGB蛋白N末端 1～ 75位氨基酸可与NKA1b2结合 ,但结合力很弱 ,而其C末端 75个氨基酸则与NKA1b2无结合作用 ,由此推测NGB蛋白整体的三维结构是结合所必需的。The pre-transformed human fetal brain cDNA library was used to screen the protein interacting with neuroglobin by using yeast two hybrid system III from ClonTech Inc.. The protein encoded by one of the clones interacting with neuroglobin (NGB) was confirmed to be the C terminus of the Na+, K+-ATPase beta2 subunit (NKA1b2) based on amino acid sequences. Then the full-length coding region cDNA sequence of NKA1b2 was obtained from human fetal brain cDNA library by PCR. A set of experiments were designed to test the interaction between NGB and NKA1b2. Interaction between NGB and NKA1b2 was confirmed by binding assay in vitro . Furthermore, the interaction was also proved by co-immunoprecipitation test in vivo . Moreover, the structure integrity of neuroglobin was found to be essential for the interaction between NGB and NKA1b2 by yeast two hybrid method with a series of neuroglobin truncated mutants.

关 键 词：脑红蛋白 NA^+ K^+-ATP酶 Β2亚基 作用位点 酵母双杂交系统 蛋白质相互作用 免疫共沉淀 

分 类 号：Q51[生物学—生物化学] Q71