Zimmerman U J, Hennigan B B, Liu L, Campbell C H, Fisher A B
Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Biochem Mol Biol Int. 1995 Feb;35(2):307-15.
We have isolated choline binding proteins from the plasma membrane fraction fraction of human lung epithelium-derived cell line (A549) by means of detergent solubilization, anion exchange and affinity chromatography. One of the affinity purified proteins had a specific choline binding activity of 44-57 pmol/mg, representing a two to three hundredfold enrichment relative to the specific activity of freshly prepared plasma membranes. The purified protein has a molecular mass of 38 kDa by SDS PAGE analysis and was identified as annexin II by N-terminal microsequencing. Annexin II, however, had not previously been known for choline binding activity. We therefore prepared a mixture of authentic annexins (I-V) from A549 cells. The mixture had a choline binding activity of 15 to 18 pmol/mg. The annexin mixture was subsequently affinity chromatographed on the choline-conjugated Sepharose 6B column. Analyses by SDS PAGE and immunoblot revealed that annexins I, II, and III are bound to the choline column while annexins IV and V did not. These results indicate that some of the annexins have specific choline binding activities.
我们通过去污剂增溶、阴离子交换和亲和层析从人肺上皮来源的细胞系（A549）的质膜部分中分离出胆碱结合蛋白。其中一种亲和纯化的蛋白具有44 - 57 pmol/mg的特异性胆碱结合活性，相对于新鲜制备的质膜的比活性，富集了200至300倍。通过SDS - PAGE分析，纯化的蛋白分子量为38 kDa，经N端微测序鉴定为膜联蛋白II。然而，膜联蛋白II以前并不以胆碱结合活性而为人所知。因此，我们从A549细胞中制备了一份包含真实膜联蛋白（I - V）的混合物。该混合物具有15至18 pmol/mg的胆碱结合活性。随后，将膜联蛋白混合物在胆碱偶联的琼脂糖6B柱上进行亲和层析。SDS - PAGE和免疫印迹分析表明，膜联蛋白I、II和III与胆碱柱结合，而膜联蛋白IV和V则不结合。这些结果表明，某些膜联蛋白具有特异性胆碱结合活性。
