摘要
目的 从牛心肌中提取纯化钙调蛋白依赖性蛋白激酶Ⅱ (CaMKⅡ )，并测定其活性。方法 采用 DEAE- cellulose柱层析和 Sepharose 4B亲和层析分离纯化蛋白，用γ- 32P掺入法测定 CaMKⅡ及自磷酸化的 CaMKⅡ活性。 结果从牛心肌中提取到钙调蛋白结合的蛋白组分，纯化后得到的 CaMKⅡ比较稳定，产量也较高。此过程比较简单，适合大量制备。经 SDS- PAGE检测，发现酶提取物均质，酶亚基的目的蛋白带相对分子质量为 56 000。在 syntide- 2为底物的反应中，纯化的 CaMKⅡ的特异性酶活性为 7.84 pmol· min－ 1· mg－ 1。 CaMKⅡ可自磷酸化，经过自磷酸化的酶在 Ca2＋和钙调蛋白不存在时也保持活性。结论 通过凝胶层析与亲和层析可提纯 CaMKⅡ。
Objective To purify the calmodulin- dependent protein kinaseⅡ (CaMKⅡ )from cardiac muscle of bovine and assay its activity. Methods CaMKⅡ was isolated and purificated by DEAE- cellulose chromatography and Sepharose 4B affinity chromatography. CaMKⅡ activity and autophosphorylated CaMKⅡ activity were determined byγ- 32P incorporation. Results CaMKⅡ was obtained with apparent homogeneity and high yield from the total calmodulin- binding protein fraction cardiac muscle of bovine. This procedure was simple and suitable for large scale preparation. Enzyme appeared to be homogeneous when examined by SDS- PAGE. SDS- PAGE of the enzyme subunit showed protein band with relative molecular mass of 56 000. The purified CaMKⅡ had a specific enzymic activity of 7.84 pmol· min－ 1· mg－ 1 protein when mixed syntide- 2 was used as a substrate. CaMKⅡ underwent autophosphorylation, the autophosphorylated enzyme remained active in the absence of Ca2＋ and calmodulin. Conclusion CaMKⅡ can be purified by gel filtration and affinity chromatography.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2001年第3期228-231,共4页
Acta Academiae Medicinae Sinicae
基金
国家自然科学基金( 39770852)资助&&
