Wu V Y, Cohen M P
University City Science Center, Philadelphia, PA 19104.
Biochem Biophys Res Commun. 1993 Jun 30;193(3):1131-6. doi: 10.1006/bbrc.1993.1743.
The ability of glycated serum proteins, which exist in vivo predominantly as Amadori adducts, to influence cell biology suggests the existence of cell binding proteins that recognize glucose adducts in nonenzymatically glycated proteins. To explore this possibility, we applied detergent extracts of aortic endothelial cells to HPLC and to an affinity column of glycated human albumin immobilized onto Sepharose, which was eluted with alkaline pH. HPLC fractionation revealed a peak reactive in ELISA with glycated but not nonglycated albumin. Two polypeptides of approximately 110 kDa and 205 kDa were identified when the affinity bound proteins were electrophoresed, transferred to immunoblotting membranes, and reacted with an enzyme conjugate of glycated albumin containing Amadori adducts. These polypeptides did not react with enzyme conjugate of carbohydrate-free albumin, confirming their binding specificity for glucose modified albumin and absence of co-identity with previously described albumin-binding proteins. The molecular weights of the glycated albumin binding polypeptides are distinct from those described for bovine albumin modified by advanced glycation end products or by formaldehyde, indicating that Amadori adducts in glycated albumin are recognized by unique endothelial cell binding sites.
体内主要以阿马多里加合物形式存在的糖化血清蛋白影响细胞生物学的能力表明，存在能够识别非酶糖化蛋白中葡萄糖加合物的细胞结合蛋白。为了探究这种可能性，我们将主动脉内皮细胞的去污剂提取物应用于高效液相色谱（HPLC）以及固定在琼脂糖上的糖化人白蛋白亲和柱，并通过碱性pH值洗脱。HPLC分级分离显示，在酶联免疫吸附测定（ELISA）中，一个峰与糖化白蛋白而非非糖化白蛋白发生反应。当亲和结合蛋白进行电泳、转移至免疫印迹膜并与含有阿马多里加合物的糖化白蛋白酶偶联物反应时，鉴定出了两条分子量约为110 kDa和205 kDa的多肽。这些多肽不与无糖化白蛋白的酶偶联物反应，证实了它们对葡萄糖修饰白蛋白的结合特异性，且与先前描述的白蛋白结合蛋白不存在共同特征。糖化白蛋白结合多肽的分子量与经晚期糖基化终产物或甲醛修饰的牛白蛋白所描述的分子量不同，表明糖化白蛋白中的阿马多里加合物被独特的内皮细胞结合位点所识别。
